Devices for conducting specific binding assays

ABSTRACT

Methods and devices for separating bound label from unbound label within an assay mixture and for predispensing assay reactants in self-contained assay vessels, as well as a method for detecting the presence and/or amount of an analyte within a fluid sample, and a reusable detection vessel for use therein and with specific binding assays in general are disclosed. Significant to the separation of bound label from unbound label is the use of a cushion comprising generally one primary layer and in some cases one or more secondary layers.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. Pat. ApplicationSer. No. 07/017,318, filed Feb. 20, 1987, now U.S. Pat. No. 4,868,130issued Sept. 19, 1989 which is a continuation-in-part to U.S. Pat.Application Ser. No. 06/768,108, filed Aug. 21, 1985 now abandoned.

TECHNICAL FIELD

This invention relates generally to specific binding assays inself-contained assay vessels, and more particularly, to methods forstoring and mixing reactants, and for separating labeled componentsbound to a solid phase from unbound labeled components in bindingassays, followed by measurement of the bound labeled components. Thisinvention also relates to methods for detecting the presence and/oramount of an analyte within a fluid sample using either a homogeneous orheterogeneous binding assay performed in a self-contained assay vessel,where the assay vessel contains a reaction mixture and a cushion whichare predispensed in one or more layers.

BACKGROUND ART

Specific binding assays have found widespread application in the fieldsof biomedical research and clinical diagnostics where they are used todetermined the presence or encountered of a variety of substances(analytes) commonly encountered in biological fluids. Such substancesmay include proteins, drugs, hormones, vitamins, microorganisms, etc. Inaddition, specific binding assays may find utility in other fields, suchas food processing and environmental quality control, for the detectionof microorganisms and their toxins, or for detecting organic wastes.

Specific binding assays are commonly divided into homogeneous andheterogeneous assays. In a homogeneous assay, the signal emitted by thebound labeled component is different from the signal emitted by theunbound labeled component. Hence, the two can be distinguished withoutthe need for a physical separation step. The classical homogeneousspecific binding assay is the enzyme-multiplied immunoassay technique(EMIT), described in U.S. Pat. No. 3,817,837, issued to Rubenstein.

Homogeneous specific binding assays are rapid and easy to perform,either manually or with automated instruments. However, these teststypically require sequential additions and mixing, with interveningincubations, of sample plus antibody, then enzyme-analyte conjugate,followed by enzyme substrate color developer solution. Automation hasbeen achieved with various types of analyzers including discrete (e.g.DuPont aca™), centrifugal (e.g. Roche Cobas Bio™), and linear flow (e.g.Technicon SMAC™) However, homogeneous assays have several disadvantages:they are typically limited to detection of low molecular weightcompounds, are prone to interferences, and are generally limited insensitivity to detection of approximately 1 nanomolar analyte.

In heterogeneous assays, both large and small analytes can be detected,but the signal emitted by the bound and unbound labeled components isidentical, hence the two must be physically separated in order todistinguish between them. The classical heterogeneous specific bindingassay is the competitive radioimmunoassay (RIA), described by Yalow(Science 200:1245, 1978). Other heterogeneous binding assays are theradioreceptor assay, described by Cuatrecasas (Ann. Rev. Biochem. 43:109-214, 1974), and the sandwich radioimmunoassay, described by Wide(pp. 199-206 of Radioimmunoassay Methods, Edited by Kirkham and Hunter,E. & S. Livingstone, Edinburgh, 1970). Because interferences are usuallyeliminated, and because excess binding reagents can sometimes be used,heterogeneous binding assays can be significantly more sensitive andreliable than homogeneous assays.

In a typical "double antibody" competitive RIA, a known amount ofradiolabeled ligand and ligand present in the sample compete for alimited amount of antibody. Sufficient time is allowed for specificbinding to occur, after which the antibody and bound ligand areprecipitated by addition of anti-immunoglobulin, washed to removeunbound label by repeated centrifugation, and the amount of labeledligand present in the bound phase is determined.

A sandwich assay can be used to achieve greater sensitivity for analytessuch as antigen in an immunoassay. In such an assay, excess ligands areused to force binding at concentrations below the dissociation constantof the binding pair. In the typical sandwich immunoassay, two antibodytypes are required, each of which can bind simultaneously to theantigen. One antibody is bound to a solid phase, while the other islabelled. As with competitive RIAs, one or more discrete washing stepsto separate bound and unbound label are required, and sequentialaddition of reagents is typical.

Because the solid phase must be isolated and washed, and becausesequential reagent additions are frequently required, heterogeneousassays tend to be time consuming and labor-intensive. However, they workequally as well for low and high molecular weight compounds, are lessprone to interferences than homogeneous assays, and can be sensitive tosub-picomolar antigen concentrations. Automation of heterogeneousimmunoassays has been accomplished (ARIA II™ by Becton Dickinson,CentRIA™ by Union Carbide), but this has required either sophisticatedand expensive instrumentation to carefully control liquid flow and tomonitor bound and unbound fractions, or it has resulted in the detectiononly of the unbound label flowing through a rapidly hydrated antibodysolid phase.

Several attempts have been made to eliminate the inconvenience ofwashing steps in heterogeneous binding assays. For example, Glover etal., GB 1,411,382, describe a method for measuring the amount of unboundradiolabel, after partial separation from bound label, by shielding thebound (lower) phase. However, it is well known in the art that thesensitivity and precision of specific binding assays is severely limitedif changes in the unbound rather than the bound labeled component aremeasured. Furthermore, methods which lack a washing step have thedisadvantage of detecting both tightly binding (specific) and weaklybinding (nonspecific) label, resulting in very high nonspecific signal.Charlton et al., U.S. Pat. No. 4,106,907, issued Aug. 15, 1978, discloseanother container for radioactive counting which consists of a taperedreaction tube having a radiation shield extending up from the bottom ofthe tube to a uniform height, such that only radiation from thesupernatant (the unbound labeled fraction) can be detected. This methodis subject to the same limitations as Glover et al., supra.

Chantot et al., Analyt. Biochem. 84:256, 1978, describe a radioreceptorassay method for measuring the binding of radiolabeled ligands tomembrane receptors. The technique involves counting the total amount ofradiolabel present, centrifuging the sample, and recounting with anexternally mounted copper screen which serves to absorb radiation from adefined volume of the supernatant. The screen itself consists of acopper sleeve mounted on the outside of a custom-made test tube having asmall knob precisely positioned above the base to support the screen.This method suffers from the disadvantage of requiring double detection,and suffers as well from high nonspecific binding as described above forthe Glover and Charlton methods. Furthermore, the tube is vulnerable tojamming and breakage in standard gamma counters. As with theabove-described "screening" methods, the large diameter of the screenallows significant scattered radiation from within the screened volumeto impinge on the detector, resulting in inaccurate measurements of theunscreened label. Also, because bound label is directly adjacent to andin contact with unbound label, normal and unavoidable variability in theposition of the screen or in the volumes of the unbound and bound phasescan cause significant variability in signal.

Bennett et al., (J. Biol. Chem. 252: 2753, 1977) describe aradioreceptor assay in which, after mixing and incubating reagents, theassay mixture is transferred to a centrifuge tube to wash the solidphase containing bound label. They employed prolonged (30minutes) highspeed centrifugation to force the solid phase into a solution of 20%sucrose, followed immediately by freezing the assay tube in liquidnitrogen and excising the tip of the tube containing the solid phase andbound label. This method provides more effective separation of bound andunbound label than those described above, but has several significantdisadvantages. The assay mixture cannot be incubated in situ on top ofthe sucrose solution, thus requiring separate incubation and separationvessels, because reactants would diffuse into the solution. Care must beused in loading the assay mixtures onto these sucrose solutions becausemixing will cause dilution of the assay mixture, thus changing theequilibrium for assay reactants. The separation is relatively lengthy,and assay tubes must be frozen immediately after centrifugation becausethe bound label can dissociate from the solid phase and diffuse awayfrom the tip of the separation tube. Finally, excising the tip ofseparation tubes is inconvenient, time-consuming, difficult to performreproducibly, exposes the user to the risk of liquid nitrogen burns andradioactive contamination from fragments of frozen tubes and theircontents, and would be very difficult to automate.

In U.S Pat. No. 4,125,375 (issued Nov. 14, 1978), Hunter describes amethod and automated instrumentation for performing heterogeneousimmunoassays by carefully injecting a sucrose solution underneath apreviously equilibrated immunoassay mixture containing particles ofhigher density than the sucrose solution. The particles are allowed tosettle through the injected subphase, thereby separating the particlesfrom the unbound label. This method potentially eliminates some of thedisadvantages inherent in the Bennett et al. method, but suffers fromseveral significant shortcomings. These shortcomings include that: (1)it requires separate preequilibration of the assay mixture prior toseparation of bound and free label, plus removal of liquid waste, andthus cannot be self-contained, (2) the method is not readily adaptableto the most rapid (centrifugal) separations, (3) it suffers frompotential dilution and diffusion artifacts as in the Bennett et al.method, (4) it is not suitable for convenient and reproducible manualassays, and (5) any automated instrument would require plumbing forreagent delivery and waste disposal.

Linsley et al., Proc. Natl. Acad. Sci. (USA) 82: 356, 1985, describe aradioimmunoassay for type I transforming growth factor using S. aureusin which the bound label is separated from the unbound label by rapidsedimentation into a solution of 10% sucrose, followed by freezing inliquid nitrogen and excision of the tip of the centrifuge tube todetermine the sedimented bound label. This method is essentially animmunoassay embodiment of the radio-receptor assay described by Bennettet al., with the inherent disadvantages of the former method.

Although each of the methods described above have brought minorimprovements to the state of the art, there remains a need in the artfor a method of specific binding assay which combines the ease andrapidity of homogeneous techniques with the enhanced sensitivity typicalof heterogeneous techniques, for both isotopic and nonisotopicapplications, without the undesirable variability, delay, labor, anddissociation which occur during the wash steps. Further, the methodshould allow rapid separations, should be convenient for manual use withstandard detection instruments, and should be readily adaptable tosemiautomated or fully automated instrumentation. Ideally the methodshould be self-contained, have minimal plumbing and moving parts, and becompatible with fully predispensed reagents. The present inventionfulfills this need, and further provides other related advantages.

DISCLOSURE OF THE INVENTION

Briefly stated, the present invention discloses methods and associateddevices for separating bound label from unbound label within a bindingassay mixture, and for predispensing a cushion in one or more liquid orsolid layers, as well as a reaction mixture, which, with the addition ofsample, forms a self-contained system for both heterogeneous andhomogeneous binding assays. In additon, methods for detecting thepresence and/or amount of an analyte within a fluid sample, assayvessels, and a reusable detection vessel for use therein and withinspecific binding assays in general are disclosed. For purposes of thepresent invention, the term "cushion" is defined to include all primaryand secondary layers within any one embodiment.

In one aspect of the present invention, a method is disclosed forseparating bound label from unbound label within an assay mixture formedwithin an assay vessel. For purposes of the present invention, thephrase "assay mixture" includes a reaction mixture, having at leastlabel and binding components, and sample. The assay mixture can be inthe form of one or more layers in an assay vessel, a layer being in theform of a droplet, or varying from a thin film to several centimetersthick depending on the volume of the assay mixture and the dimensions ofthe assay vessel. The assay mixture generally comprises a reagentmixture plus a sample containing analyte. The reagent mixture furthercomprises one or more binding components and may further comprise one ormore labels. Binding components normally comprise two parts: a solidphase and a specific binding agent attached thereto, which confersspecific binding activity. Additional specific binding agents may bepresent which are not initially attached to the solid phase, as long assubstantially all of the additional binding agent becomes attached tothe solid phase prior to separation of bound label from unbound label.In addition, other binding agents may be added subsequent to separationof bound label from unbound label.

Once the reagent mixture and sample are combined to form the assaymixture, an incubation period is usually required. The incubation periodcan range from one second to several days, depending in part uponfactors such as the sensitivity required, and the binding affinity andconcentration of binding components. Following incubation of the assaymixture, at least some label and/or analyte is bound to at least some ofthe binding components. The incubated assay mixture typically includessome unbound label and/or unbound analyte and in addition also includesother components such as water, buffer, preservative, and proteins,these components typically comprising a largely aqueous solution.

As an alternative to forming the complete assay mixture within an assayvessel, an assay mixture may be prepared outside of the assay vessel.This alternative is appropriate for non-isotopic binding assays, therebyavoiding the potential hazards associated with handling of radioisotopesoutside of the assay vessel. This alternative is especiallyadvantageous, for instance, when automated liquid-handling apparatus isavailable to the user for dispensing reagents

Briefly, these methods comprise (a) contacting a primary layer with anassay mixture, both the binding components and the unbound label beingimmiscible with the primary layer and the binding components being of adifferent density than the primary layer; and (b) subjecting the assaymixture in contact with the primary layer to conditions sufficient tocause the binding components and the unbound label to separate.Typically the binding components have a density greater than that of theprimary layer and the aqueous solution component of the assay mixturehas a density less than or equal to that of the primary layer. In someembodiments, barrier layer (preferably selectively liquifiable) ispositioned between the assay mixture and the primary layer. The barrierlayer, when liquified, is miscible with the assay mixture, while in thesolid or gel form, it serves to separate the reaction mixture from theprimary layer. A barrier layer is especially useful when predispensingof reagents is desired with an embodiment utilizing a liquid primarylayer.

In particular embodiments, either or both the binding components and theunbound label may be of the same density as the primary layer. In onesuch embodiment, the binding components are formed by immobilizingspecific binding agents to the surface of a vessel containing theremainder of the assay mixture, and thus the density of the bindingcomponents is not relevant to the assay. In another such embodiment, thebinding components are magnetic particles and are separated from unboundlabel by magnetic forces. In such cases, the binding components need notdiffer significantly in density from the primary layer, though typicallythe aqueous solution component of the assay mixture will have a densityless than that of the primary layer.

In additional embodiments, which are typically homogeneous bindingassays, the density of the entire assay mixture may be greater than thedensity of the primary layer. One such embodiment utilizes a barrierlayer or a selectively liquifiable primary layer which is in a solidform during the addition of sample and incubation, and is then liquifiedto allow mixing of the assay mixture with a secondary layer containingadditional reagents such as enzyme substrate color developer.

In a related embodiment of the present invention which includes thepredispensing of the cushion and the reaction mixture, the reactionmixture is contacted with the primary layer, as described above,substantially prior to the addition of sample and the subsequentincubation of the assay mixture. For both heterogeneous and homogeneousbinding assays, this provides advantages to the user of greaterconvenience compared to assays where each reactant must be dispensed asneeded. Furthermore, where precise and automated equipment is used topredispense the assay reactants during manufacture of the assay system,greater precision is to be expected compared to manual dispensing ofreactants by the user as they are needed.

In some embodiments, one or more reactants are contained separately fromthe reaction mixture in the assay vessel For example, in a competitiveimmunoassay, label and binding components, specifically antibody-capturebinding components, may be predispensed to form a reaction mixture.Following addition of sample to the reaction mixture, the reaction isinitiated by addition of analyte-specific antibody. Thisanalyte-specific antibody, which is typically a solution, can becontained in a reagent reservoir prior to initiation of the reaction.For example, as will be discussed more fully below, a secondary layer inthe cushion can serve as a reagent reservoir containing additionalreaction components. Alternatively, a hollow cap having a removable sealmay be provided to serve as a reagent reservoir. One kind of seal couldbe fashioned by providing a small orifice in the hollow cap, where theorifice is plugged with a selectively liquifiable material (preferablywater-immiscible). A moderate force such a air pressure or low-speedcentrifugation could be employed to force the liquid from the capreservoir into the reaction mixture.

In a related aspect of the present invention, a method is disclosed fordetecting the presence and/or amount of an analyte within a fluid sampleusing either a homogeneous or heterogeneous binding assay performed in aself-contained assay vessel. The assay vessel typically contains assayreactants which are predispensed in one or more layers. In someembodiments for detecting the presence or amount of analyte within afluid sample, the label may comprise the analyte itself, where theanalyte is capable of emitting a detectable signal. Such analytesinclude hemoglobin as well as enzymes (prostatic acid phosphatase,creatine kinase)

For example, an assay for detecting the percentage of glycosylatedhemoglobin present in blood typically involves separating most or all ofthis analyte from a blood sample using a binding component in the formof an ion exchange or affinity column, then measuring the absorbance ofthe bound analyte (glycosylated hemoglobin) as well as the absorbance ofthe unbound label (which includes nonglycosylated hemoglobin) using asuitable colorimeter. In this embodiment, the same particles used incommercially available columns (Pierce, Rockford, Ill.) can be used asbinding components in the present invention. After a suitableincubation, the assay mixture is subjected to conditions sufficient tocause the binding components and the unbound label to separate and thebound label (analyte) is detected Preferably both bound and unboundlabel are measured to allow the calculation of the percentage of analytewhich is bound. The bound label may be eluted from the particles (suchas with a sugar solution for glycosylated hemoglobin), prior to theabsorbance measurement.

In an additional related aspect of the present invention, severaldevices for separating bound label from unbound label within an assaymixture as described above are disclosed. In one embodiment, the devicecomprises an assay vessel having an open proximal end, preferablyresealable, and a closed distal end, the vessel defining an elongatedchamber therewithin. In another embodiment, the device comprises amultiwell plate. In a further embodiment, the device comprises elongatedassay vessels or strips of connected elongated assay vessels. The assayvessels are typically positioned such that they have substantially thesame spacing as the wells in a multiwell plate. These devices have aprimary layer which most often extends generally transversely within thechamber or across the well to form a selective barrier therein, theprimary layer being immiscible with both the binding components and theunbound label, and typically of different density than the bindingcomponents. For narrow, elongated vessels, the orientation can bevertical, horizontal, or intermediate between the two extremes withoutmixing any liquid layers. The assay vessel may also contain a barrierlayer positioned between the proximal end and the primary layer.

In another aspect of the present invention, an alternative method isdisclosed for detecting the presence or amount of an analyte within afluid sample. Briefly, the method comprises (a) incubating the fluidsample with a reagent mixture to form an assay mixture, the assaymixture being formed within an assay vessel, with the assay mixturecontaining one or more binding components, label, analyte, and othercomponents, and with at least some of the label and some of the analytebinding, directly or indirectly, to the binding components; (b)contacting a primary layer with the assay mixture, the bindingcomponents having label and/or analyte bound thereto, and the unboundlabel being immiscible with the primary layer; (c) subjecting the assaymixture in contact with the primary layer to conditions sufficient tocause the binding components and the unbound label to separate; and (d)detecting the label bound to the binding components and therefromdetermining the presence or amount of the analyte.

A particularly preferred embodiment of the method disclosed abovecomprises contacting the primary layer with the fluid sample and reagentmixture prior to incubation of the resultant assay mixture. Within thisembodiment, the formation and incubation of the assay mixture occurs inthe assay vessel in which the separation is carried out.

An additional preferred embodiment of the method disclosed abovecomprises including, in one or more secondary layers, supplementaryassay components which are preferably added to the binding componentsafter bound label is separated from unbound label. Supplementary assaycomponents may include label such as an enzyme-conjugated antibody,specific binding agent such as unconjugated antibody, enzyme substratecolor developer, and enzymes such as proteases. Other substancescontained in secondary layers such as those listed in Table 2 may beconsidered, in some cases, to be supplementary assay components if theyperform an additional function beyond adjusting the density of thesecondary layer solution.

An additional aspect of the present invention discloses a reusabledetection vessel for use in specific binding assays which useradioactive labels. The detection vessel generally comprises anelongated container having an open end and a closed end, and a radiationshield adapted to fit within the elongated container and positionedtherein to provide a shielded portion, and an unshielded portion towardthe closed end. In most instances it is preferable to use a shield whichhas a substantially cylindrical bore, which better provides effectiveand uniform shielding. Although not essential, it may be convenient toprovide the shield with a cylindrical exterior. In one embodiment, thisdesign allows a portion of an assay vessel, which has been inserted intothe detection vessel, to protrude downward from the shield a distancesufficient to allow detection of the label within the exposed portion ofthe assay vessel. In another embodiment, the detection vessel isprovided with a substantially cylindrical member positioned between theshield and the distal end of the container, the cylindrical member beingadapted to support and maintain the position of the shield within thecontainer. In still another embodiment, the cylindrical member is closedat the distal end to support an additional thin radiation shield in theform of a disk. The disk allows more effective shielding when usingcertain detection instruments such as certain well-type gamma counters.

Other aspects of the present invention will become evident uponreference to the following detailed description and attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a side elevation view of an assay vessel and related closuresof the present invention.

FIG. 1B is a side elevational view of an alternative assay vessel of thepresent invention.

FIG. 2 is a fragmentary side elevational view of a multi-well plateassay vessel of the present invention.

FIG. 3 is a side elevational view of a reusable detection vessel of thepresent invention, with an assay vessel placed therein.

BEST MODE FOR CARRYING OUT THE INVENTION

The following terms are defined herein for clarity:

REACTION COMPONENT: a substance or reagent which participates in and isa component of the binding assay; typically one of several solvents,solutes, or solids which are mixed to form a reaction mixture, and/orwith sample to form an assay mixture. A reaction component also may bestored within a reagent reservoir.

REAGENT RESERVOIR: an enclosure, matrix, or device in the assay vesselin which reaction components are predispensed or stored, separate fromthe reaction mixture or the assay mixture. A reagent reservoir may belocated distal to the primary or a secondary later, relative to theproximal end of the assay vessel, or within a secondary layer.Alternatively, it may be located in or adjacent to the proximal end ofthe assay vessel.

REACTION MIXTURE: the primary solution or suspension of reactioncomponents which, upon addition of sample, becomes the assay mixture.The reaction mixture is typically in contact with or in proximity to theprimary layer, and includes at least label and binding components.

ASSAY MIXTURE: the combination of a reaction mixture with a sampleresults in the formation of an assay mixture. Other reaction componentsmay be subsequently added.

BINDING COMPONENTS: include specific binding agents (such as antibody)and a solid phase (nascent or preformed, particulate or a continuoussurface).

CUSHION: includes the primary and any secondary layer(s), as well as anyassay components which are not in the reaction mixture or assay mixture,except those that are contained in one or more discrete reagentreservoirs.

PRIMARY LAYER: a substance which is not miscible with water, and extendsgenerally transversely within an assay vessel (typically in contact withthe assay mixture), and serves to separate bound from unbound label byallowing the penetration of binding components without allowing thepenetration of unbound label. Generally, the primary layer is a liquidin the temperature range of 15°-50° C. Preferred are primary layerswhich are selectively liquifiable.

SECONDARY LAYER: any layer or material distal from the primary layerrelative to the assay reaction mixture, assay mixture, or proximal endof the assay vessel. A secondary layer may be water miscible orimmiscible, and may comprise a solid or liquid. Typically a secondarylayer is in a liquid form in the range of 15°-50° C. Desirable secondarylayers are selectively liquifiable.

BARRIER LAYER: located between a reaction or assay mixture and theprimary layer, the barrier layer serves to prevent contact and/or mixingbetween the mixture(s) and the primary layer, is selectivelyliquifiable, and in its liquid form is miscible with the aqueoussolution of the reaction mixture.

SELECTIVELY LIQUIFIABLE: A substance is selectively liquifiable when itcan be reversibly converted to a liquid from a solid or gel. Generallythis is accomplished by melting, in the temperature range of 15°-50° C.

As noted above, heterogeneous specific binding assays are typically moresensitive than homogeneous assays. However, in practice this advantageis often outweighed by the labor-intensive and time consumingmanipulations of the assay mixture which are typically required. Evenwith homogeneous assays, several separate, sequential additions of assayreagents are often required. The present invention is concerned withmaterials and methods for the performance of more convenient and lesslabor-intensive specific binding assays, including both homogeneous andheterogeneous assays. Such binding assays can be performed manually orwith automated instruments designed to perform homogeneous orheterogeneous assays. In heterogeneous assays, a binding component isemployed which comprises a solid phase and attached specific bindingagent, and typically binds at least some of the label to produce bothbound and unbound label. In homogeneous assays, a binding agent isemployed which is typically dissolved in solution.

An important advantage for heterogeneous assays provided by the presentinvention is that either the reagent mixture or the assay mixture (whichincludes sample) can be stored or incubated in contact with a barrierlayer or a primary layer. Such storage of the reagent mixture isadvantageous because it allows the reaction components and cushion to beprepackaged. This reduces the number of manipulations by the user inpreparing for and performing the assay, and can improve bothconvenience, speed, and precision. Because separate storage of washbuffer as well as collection and disposal of waste liquids areeliminated, the present invention reduces the space requirements andincreases the safety of laboratory testing. Furthermore, field testingis facilitated because no water is required.

Another important advantage provided by the present invention which isrelevant to both homogeneous and heterogeneous enzyme-labeled assays isthat supplementary assay components can be predispensed in one or morelayers separate from the assay mixture layer, to create a completelyself-contained assay vessel for determining the presence and/or level ofan analyte. In the prior art, such supplementary assay components (forexample, enzyme substrate color developer for homogeneous assays, andlabeled antibody in sandwich immunoassays) typically are added after anincubation step and in some cases after the separation of bound fromunbound label and/or analyte. There exists significant commercialadvantage in the present invention for predispensing all assay reactantsso that such features as user convenience as increased compared to theprior art.

In another aspect, the invention is concerned with methods and devicesfor selectively measuring bound label after the separation has beenperformed. In some embodiments, measurement of bound label isfacilitated by shielding the unbound label from the detector.

A. BARRIER LAYERS

The barrier layer serves to prevent contact and/or mixing between themixture(s) and the primary layer, typically is selectively liquifiable,and in its liquid form is miscible with the aqueous solution of thereaction mixture. It is located between a reaction or assay mixture andthe primary layer. The preferred barrier layer is an agarose or gelatingel which can be melted in the temperature range of 15°-50° C. Barrierlayers for other applications have been described in U.S. Pat. No.4,522,786 (issued June 11, 1985, to R. C. Ebersole), which is hereinincorporated by reference.

B. PRIMARY AND SECONDARY LAYERS OF THE CUSHION

The methods of this invention generally employ a largely aqueous assaymixture containing a binding component and label, and a primary layer. Aprimary layer is immiscible with water and extends generallytransversely within an assay vessel (typically in contact with the assaymixture). Generally, the primary layer is a liquid in the temperaturerange of 15°-50° C. Preferred are primary layers which are selectivelyliquifiable. One or more secondary layers may also be employed.

The primary layer serves at least one of two functions depending onwhether the binding assay is homogeneous or heterogeneous. In aheterogeneous assay, the primary layer serves to separate bound fromunbound label by allowing the penetration of binding components withoutallowing the penetration of unbound label. In a homogeneous assay, theprimary layer separates the reaction and assay mixtures from otherreaction components which must be added to the assay mixture after aninitial incubation step. Under suitable conditions, the assay mixturepasses through the primary layer to contact or mix with other reactioncomponents such as enzyme substrate color developer.

In certain cases a composite primary layer can be produced which hascertain surprising advantages. For example, a primary layer of diphenylmethane (DPM) is selectively liquifiable, with a melting temperature ofapproximately 26° C. However, the density of liquid DPM is almost thesame as water (1.001 at 26° C.) and less dense than many reactionmixtures or assay mixtures. Thus for such cases, the aqueous mixtureitself may pass through the primary layer after it is liquified bywarming, without causing the separation of binding components fromunbound label.

However, solidified DPM can be exploited by combining it with liquidprimary layer material of different (typically greater) density, to forma composite primary layer. For example, if DPM is layered on a denseaqueous secondary layer and chilled to solidify the DPM, then a liquidprimary layer material such as diethylmethylmalonate (d=1.013) can belayered on top of the DPM. This forms a composite primary layer withboth liquid ("high density") and solid ("low density") components. Whenreagents or sample is pipetted into the vessel, the solid layer at thebottom of the composite primary layer prevents mixing of the reagentsand/or sample with the secondary layer(s). For this mixture, warming atroom temperature (20°-22° C.) for 15-20 minutes results in liquificationof the primary layer.

The barrier layer, if present, and the cushion are in a liquid form, atleast during the separation of bound from unbound label by the primarylayer and during any subsequent steps in which binding componentspenetrate additional layers. The primary layer is also largely ortotally immiscible with the assay mixture. These two features alloweffective contact of the binding components with the primary layer, withthe concomitant exclusion of the aqueous components of the assaymixture. In many embodiments, the primary and secondary layers may alsobe of a density different than the assay mixture (typically thedensities are greater than that of the assay mixture), so that therelative positions of the assay mixture and the cushion layers can bemaintained under the forces of gravity or centrifugation.

Separated from the assay mixture layer by the primary layer, one or moreadditional layers may be employed which may be miscible or immisciblewith aqueous solutions. These additional layers are hereinafter referredto as "secondary layers". Each secondary layer typically is of differentdensity than the other layers employed, and in addition is largely ortotally immiscible with any adjacent layers. In general, all layersshould be resistant to mixing or inversion, or should return to theirrelative positions on brief standing. This can be achieved by selectingat least one layer material which is a solid at the temperature ofstorage, or by using layers which differ greatly in density and areimmiscible (e.g. butyl phthalate and fluorocarbon oil). Misciblemixtures of materials such as those materials listed in Table 1 alsohave useful properties. For example, by blending two or more substanceswhich melt higher than 50° C. one could obtain a eutectic mixture with adesirable density and a desirable melting temperature in the 15°-50° C.range.

Surprisingly, the inclusion of detergent in one or more layers or in theassay mixture in some cases facilitates spontaneous separation of mixedliquid layers. Suitable detergents include nonionic (such as NonidetP-40 or Triton X-100) and ionic detergents (such as taurodeoxycholate ordodecyl sulfate) and various mixtures of detergents.

The primary layer can be composed of any of a variety of compoundsprovided that it is substantially immiscible with the components of theassay mixture, and typically will have a liquid density different thanthe solid and liquid components of the assay mixture. In instances wherethe primary layer has a density equal to or greater than the aqueoussolution of the assay mixture, the density of the primary layer isusually approximately 1.01 or greater. For such instances involvingcentrifugal separations, the density of the primary layer typically doesnot exceed 1.20, and is most preferably greater than 1.03 and less than1.15. Furthermore, for heterogenous binding assays the density of theprimary layer typically will be less than the apparent density of thebinding components. In addition, the primary layer will be in a liquidform at least for the separation step or supplementary reagent mixingstep following incubation. Secondary layers also will be in a liquidform, at least for any supplementary reagent mixing steps, and/or duringsuch periods that the binding components are desired to penetrate orpass through the secondary layers. Liquification of solid primary layerstypically involves melting, generally in the range of 15°-50° C. Inapplications utilizing gravity or centrifugation to achieve separationsin heterogeneous binding assays, or where a supplementary reagent mixingstep is desirable in homogeneous binding assays, the density of theprimary and any secondary layers should be different than the density ofthe assay mixture. A representative listing of water-immiscible denseoils suitable for use as primary layers is shown in Table 1. Thesematerials may also be used as components of secondary layers.

For embodiments where the primary layer is more dense than the liquidcomponents of the assay mixture, the primary

                                      TABLE 1                                     __________________________________________________________________________    REPRESENTATIVE LIST OF WATER-IMMISCIBLE SUBSTANCES                                                       MERCK #                                                                       (9TH ED)                 SOLUBILITY                ITEM CHEMICAL NAME         OR VENDOR                                                                              DENSITY                                                                             MP/FP                                                                              Mol Wt                                                                             (parts                    __________________________________________________________________________                                                        H2O)                       1   ETHYL ACETOACETATE    3686     1.03  -45  130  35                         2   ETHYL ACETYLSALICYLATE                                                                              3687     1.15  N.A. 208  INSOL                      3   METHYL ADIPATE        ALDRICH  1.06    8  174  N.A.                       4   ETHYL ADIPATE         3689     1.01  -18  202  INSOL                      5   METHYL BENZOATE       5899     1.09  -15  136  INSOL                      6   ETHYL BENZOATE        3697     1.05  -34  150  ALMOST INSOL               7   ETHYL BENZOYLACETATE  3698     1.12  N.A. 192  INSOL                      8   ETHYL BENZENESULFONATE                                                                              3696     1.22  N.A. 186  SLIGHTLY                   9   METHYL CARBONATE      5912     1.06  0.5   90  INSOL                     10   METHYL CINNAMATE      2288     1.04   36  N.A. ALMOST INSOL              11   ETHYL CINNAMATE       2288     1.04    8  N.A. INSOL                     12   BUTYL CINNAMATE       2288     1.01  N.A. N.A. 200                       13   TRIETHYL CITRATE      3719     1.14  N.A. 276  14.5                      14   BUTYL CITRATE         1551     1.04  -20  360  INSOL                     15   DIMETHYL FUMARATE     ALDRICH  N.A.   103 -16                                                                           DIETHYL FUMARATE                                                                   ALDRICH 1.05 1-2                                                              172. N.A.                 17   METHYL FUROATE        5943     1.18  N.A. 126  SLIGHTLY                  18   DIETHYL GLUTACONATE   SIGMA    1.05  N.A. N.A. N.A.                      19   DIMETHYL GLUTARATE    4305     1.09  N.A. 160  N.A.                      20   DIETHYL GLUTARATE     ALDRICH  1.02  N.A. 188  N.A.                      21   DIMETHYL ITACONATE    ALDRICH  1.12  37-40                                                                              158  N.A.                      22   DIETHYL MALEATE       3761     1.06  -10  172  INSOL                     23   DIETHYL ACETAMIDOMALONATE                                                                           ALDRICH  N.A.   97                                 24   DIMETHYL MALONATE     5961     1.16  -62  132  SLIGHTLY                  25   DIETHYL MALONATE      3763     1.06  -50  160  50                        26   DIETHYL METHYL MALONATE                                                                             SIGMA    1.01  N.A. N.A. N.A.                      27   DIETHYL BENZYL MALONATE                                                                             ALDRICH  1.06  N.A. 250  N.A.                      28   ETHYL OXALACETATE     3776     1.13  N.A. 188  INSOL                     29   DIMETHYL OXALATE      ALDRICH  1.15  50-54                                                                              118  17                        30   DIETHYL OXALATE       3109     1.08  -41  146  SPARINGLY                 31   ETHYL PHENYLACETATE   3780     1.03  N.A. 164  N.A.                      32   DIMETHYL PHTHALATE    3244     1.19    0  194  232                       33   DIETHYL PHTHALATE     3783     1.23  N.A. 222  INSOL                     34   DIPROPYL PHTHALATE    ALDRICH  1.08  N.A. 250  N.A.                      35   DIBUTYL PHTHALATE     1575     1.04  -35  278  2500                      36   METHYL SALICYLATE     5990     1.18  -8.6 152  1500                      37   ETHYL SALICYLATE      3793     1.13    1  166  SLIGHTLY                  38   DIMETHYLDIPHENYLPOLYSILOXANE                                                                        SIGMA    1.05  N.A. N.A. INSOL                     39   SILICONE OIL          SIGMA    1.05  N.A. N.A. INSOL                     40   DIMETHYL ACETYL SUCCINATE                                                                           ALDRICH  N.A.   33                                 41   DIETHYL ACETYL SUCCINATE                                                                            ALDRICH  N.A.  N.A.                                42   DIMETHYL SUCCINATE    5993     1.12  19.5 146  120                       43   DIMETHYL METHYL SUCCINATE                                                                           ALDRICH  1.08  N.A. 160  N.A                       44   DIETHYL SUCCINATE     3799     1.04  -21  174  insol                     45   DIMETHYL L-TARTRATE   ALDRICH  1.24  48-50                                                                              178  N.A                       46   DIETHYL L-TARTRATE    3803     1.20   17  206  slightly                  47   DIBUTYL L-TARTRATE    ALDRICH  1.09  21-22                                                                              262  N.A.                      48   FLUORINERT FC-40 (3M) 3M       1.85  N.A. N.A. INSOL                     49   FLUORINERT FC-70 (3M) 3M       1.93  N.A. N.A. INSOL                     50   FLUORINERT FC-77 (3M) 3M       1.78  N.A. N.A. INSOL                     51   DIPHENYLMETHANE       3339     1.00   26  168  N.A.                      __________________________________________________________________________

layer materials will have the properties of oils with densities greaterthan water (d>1.00). However, for some homogeneous binding assaysrequiring a supplementary reagent mixing step, in which the entirereaction mixture penetrates the primary layer to mix with one or moresupplementary assay components in a secondary layer, an oil with adensity to water can be employed if it can be less than or equalmaintained in a solid form during incubation, then subsequentlyliquified. In such embodiments, the reaction mixture may contain one ormore materials which form dense aqueous solutions. A representative listof such water-miscible materials forming dense aqueous solutions isshown in Table 2.

Dense oil-like materials are typically synthetic esters (usually methyl,ethyl, propyl or butyl) of bioorganic acids, and usually containsubstantial oxygen, nitrogen, or sulfur or they are fluorocarbon oils orsilicon-based oils. Most dense oil-like materials are miscible with eachother and can be used alone or in various mixtures in primary orsecondary layers. However, in some embodiments it is possible anddesirable to create adjacent water-immiscible layers which are notmiscible with each other and which differ in density (e.g. ahydrocarbon-based material or mixture plus a fluorocarbon-based materialor mixture). In such embodiments a water-immiscible layer which is notin contact with the assay mixture would be called a secondary layer.

To those experienced in organic chemistry and others skilled in the art,related water-immiscible materials which have desirable properties,other than those materials listed in Table 1, will be readily apparent.Such properties include partial or complete immiscibility in water andaqueous solutions and lack of objectionable odor or toxicity. A furtherdesirable property of a primary layer material is the ability to rapidlyand spontaneously reform a homogeneous phase when mixed with a reagentmixture or assay mixture. Further, the primary layer must be in a liquidform during the separation step (in heterogeneous assays) and thesupplementary reagent mixing step (in homogeneous assays).

While most of the applications described for the present invention canutilize dense oils as primary layer materials, it will be apparent tothose skilled in the art that water-immiscible substances with densitiesless than water could be blended materials such as high-melting denseoils to form mixtures with useful densities and melting temperatures, orcould be used in applications where the primary layer is less dense thanthe assay mixture. Many organic solvents could be used, as well as fatsand waxes. Amphiphilic substances which would disperse in water onmelting could be useful additives to barrier layers or primary layers(Hargreaves et al., Biochemistry 17:3759-3768, 1978, herein incorportedby reference).

For water-immiscible dense oils Which are useful as solids in themethods of the present invention, liquification typically takes placewithin the range from 15° to 50° C. In some cases the temperature ofliquification for meltable water-immiscible dense oils can be controlledby blending two or more substances which individually melt at highertemperatures than the mixture of the substances. It will also beapparent to those skilled in the art that liquification can be achievedin some cases by means other than melting, such as by depolymerizationof a solid polymer.

Particularly preferred for centrifugal applications are primary layermaterials or mixtures thereof with densities in the approximate range of1.04 to 1.15, such as dipropyl or dibutyl phthalate, methyl cinnamate,ethyl cinnamate, butyl cinnamate, butyl citrate, diethyl fumarate,dimethyl itaconate, diethyl maleate, diethyl oxalate, diethyl succinate,and dibutyl tartrate. Where a detergent is used in the assay mixturewith a liquid primary layer, the preferred primary layers include butylphthalate, ethyl cinnamate, ethyl salicylate, silicon oil (Table 1,#38), and dimethyldiphenylpolysiloxane, because materials such as thesedo not form unwanted emulsions with reaction mixtures containingdetergents. Where no detergent is used, the preferred primary layermaterials include diethyl succinate, methyl adipate, dimethyl succinate,ethyl salicylate, dimethyl malonate, and diethyl malonate, because theyreadily separate into two or more clear phases when mixed with aqueousreaction mixtures that lack detergent.

Particularly preferred for embodiments in which the binding component isattached to the surface of the assay vessel are primary layers offluorocarbon oils, because of the low viscosity and high density ofthese oils, which properties aid in the complete displacement of waterfrom the binding components in such embodiments. Fluorocarbon oils arealso attractive for such applications because polystyrene assay vesselscan be used with such oils.

For other embodiments in which the assay vessel is desired to be clearplastic such as polystyrene, preferred primary layer materials includemethyl cinnamate or methyl itaconate (stored below 36° C.), silicon oil(Table 1 #38, "high temperature" melting point bath oil, from SigmaChemical Co., St. Louis, Mo., or from Aldrich Chemical Co., Milwaukee,Wis.), and dimethyldiphenylpolysiloxane. Preferred for embodiments inwhich the primary layer is desired to be in a solid form within somepart of the temperature range from 15°-50° C., are primary layerscontaining methyl cinnamate, dimethyl itaconate, dimethyl oxalate,dimethyl succinate, dimethyl, diethyl, and dibutyl tartrate, ordiphenylmethane, or mixtures of these substances. For those embodimentswhich utilize both centrifugal separations and solid primary layers inthe range of 15°-50° C., the preferred primary layer materials aremethyl cinnamate and dimethyl itaconate and mixtures of thesesubstances.

Depending on the nature of the signal emitted or produced by the label,the washing effectiveness or supplemental reaction required, it may bedesirable to include a secondary layer or layers. While a secondarylayer may be formed using an appropriate water-immiscible material fromTable 1, a secondary layer may also be water-soluble. To form watersoluble secondary layers, or to increase the density of an assay mixturefor applications such as homogeneous assays, typically a material isdissolved in water to increase its density. A representative listing ofmaterials appropriate for this purpose is shown in Table 2. Thesematerials are especially well suited for use as components of secondarylayers or assay mixtures as described above. However, in certaininstances, a material which is soluble both water and inwater-immiscible substances (e.g. formamide or dimethylsulfoxide) may beused within a primary layer. In another embodiment, formamide may beincluded in a DNA hybridization assay mixture and/or a primary layer forsuch an assay to facilitate the hybridization of polynucleotide strands.

For use with homogeneous enzyme-labeled immunoassays, an aqueoussecondary layer containing enzyme substrate may have the same or similardensity as the assay mixture. In such an embodiment, the primary layerwill typically be a solid during incubation prior to color development.For example, if the primary layer is less dense than both the assaymixture and the secondary layer, the primary layer will float to the topof the assay vessel upon melting. This will allow the assay mixture andthe substrate-containing secondary layer to merge in the bottom of theassay vessel. In this embodiment, the primary layer material can be lessdense than water if it can be solidified after dispensing onto animmiscible secondary layer of greater density. An electromagnet can beused to obtain effective mixing of the assay mixture and secondary layerafter liquification of the primary layer, if several paramagneticparticles are included in the assay vessel.

Further, it may be desirable to include supplementary assay componentsin either primary or secondary layers which aid in signal production ordetection. An example of an

                                      TABLE 2                                     __________________________________________________________________________    Representative Dense, Water-Miscible Liquids                                  CHEMICAL NAME DENSITY                                                                             CONC.                                                                              COMMENTS                                             __________________________________________________________________________    CESIUM CHLORIDE                                                                             1.174 20%                                                       CESIUM SULFATE                                                                              1.190 20%                                                       DIETHYLENE GLYCOL                                                                           1.118 100%                                                      DIMETHYLSULFOXIDE                                                                           1.100 100% MP = 18 DEGREES                                      ETHYLENE GLYCOL                                                                             1.114 100%                                                      FICOLL        1.068 20%                                                       FORMAMIDE     1.130 100% MP = 2.6 DEGREES                                     GLYCEROL      1.045 20%                                                       LITHIUM BROMIDE                                                                             1.160 20%  SOL. IN .6 PARTS H2O                                 LITHIUM CHLORIDE                                                                            1.113 20%  SOL. IN 1.3 PARTS H2O                                LITHIUM SULFATE                                                                             NA         SOL. IN 2.6 PARTS H2O                                METRIZAMIDE   1.110 20%  DENSITY AT 15 DEGREES                                PERCOLL       1.300 100% SELF-FORMING GRADIENTS                               POTASSIUM ACETATE                                                                           1.100 20%                                                       POTASSIUM BROMIDE                                                                           1.158 20%                                                       POTASSIUM CITRATE                                                                           1.140 20%                                                       POTASSIUM IODIDE                                                                            NA                                                              POTASSIUM TARTRATE                                                                          1.139 20%                                                       PROPYLENE GLYCOL                                                                            1.036 100% MISC WITH H2O, CHCL3                                 SODIUM BROMIDE                                                                              1.172 20%                                                       SORBITOL      1.079 20%  SOLUBLE TO 83%                                       SODIUM IODIDE NA                                                              SUCROSE       1.079 20%                                                       __________________________________________________________________________

additive for a primary or secondary layer is a scintillation fluor, suchas 2,5 diphenyloxazole (PPO) or 1,4-bis[5-pheny-1-2-oxozolyl]benzene(POPOP), which may be included in a primary or secondary layer if thelabel can be detected in a scintillation counter using such fluors.Additives to a secondary layer can also include enzymes, proenzymes(zymogens), or enzyme substrate, where the label is an enzyme substrate,a zymogen activator, or an enzyme, respectively. In some embodiments(e.g. certain sandwich binding assays) where a label is added to theassay mixture after an initial incubation and separation of bound fromfree analyte, a secondary layer may contain label (e.g. labelledantibody).

Secondary layers can also be formulated to contain chaotropic agents,such as salts, urea, guanidinium chloride, nonionic or ionic detergents,etc. to reduce nonspecific binding. In any case, the concentrations ofthese additives typically should not be sufficient to cause significantdissociation of specifically bound label from its binding componentduring the movement of the binding component through such layers.However, in some embodiments, dissociation of label from its bindingcomponent is desirable and can be achieved by inclusion of a suitabledissociating agent in a secondary layer. For example, sorbitol is willdissociate glycosylated hemoglobin from the boronic acid particles usedin a commercial column chromatography kit from Pierce (Rockford, Ill.)

As noted above, for the purpose of the present invention, the term"cushion" is defined to include all primary or secondary layers, aloneor used in combination. The volume of the cushion in differentembodiments is variable and will depend on a number of factors,including the particular label employed, the detection mode, therequired sensitivity of the assay, and the assay mixture volume. Boththe volume and formulation of the cushion can be determined empirically.For most isotopic applications, however, a ratio of 2.5 volumes ofcushion to one volume of sample will be adequate where it is required toshield radiation emanating from unbound label.

For multilayer cushion embodiments, and in cases where nonspecificbinding is adequately low, the volume of the primary layer need only beenough to completely isolate the assay mixture from the secondarylayer(s) under the conditions used. Where no secondary layers are used,the primary layer need only isolate binding components from the assaymixture after the separatn step. A ratio of assay:cushion volumesgreater the 1:1 can be used in some cases. Typically for competitiveassays, approximately 3-4% nonspecific binding is acceptable, while 1-2%is very good. For sandwich assays where excess label may be used,nonspecific binding may be required to be 0.2% or below. Nonspecificbinding is determined largely by the physical properties of the labeland the binding components and will vary.

For example, in the use of a 96-well plate, a ratio of one volume ofprimary layer to one volume of sample will usually be adequate. Asmaller amount of primary layer may be usable if it is in a solid formduring sample loading, or if the assay mixture is immiscible with allprimary and secondary layers.

The geometry and orientation of the assay vessel, the assay mixture, andthe cushion will be governed by particular applications. In a typicaluse involving centrifugal or gravity separation, one of many types oftest tubes or multiwelled plates can be used. In most uses, the sample,binding components, and secondary components are conveniently added,mixed, and incubated in contact with the predispensed primary layer. Insome cases, binding components and/or secondary components can bepredispensed along with the cushion in sealed assay separation vessels.In such cases, fewer components (as few as one, the sample) need beadded by the user prior to mixing and incubation.

An additional option is available with magnetic separations, wherein thecushion layer(s) can be oriented lateral to the assay mixture, or abovethe assay mixture if the density of assay mixture is greater than thatof the layer(s).

In the case where the binding components are attached to the surface ofthe assay vessel, the assay mixture can be pre-equilibrated in contactwith the binding components at the bottom of the assay vessel. Toachieve separation of bound label from unbound label, a primary layermaterial can be poured or pipetted into the assay vessel to displace theless dense secondary components (including unbound label) to the top ofthe primary layer. In some cases, secondary layers can be addedsimultaneously with or subsequent to the primary layer addition.

C. SOLID PHASES USED IN BINDING COMPONENTS

Binding components normally comprise two parts: a solid phase and aspecific binding agent attached thereto, which confers specific bindingactivity. Several types of solid phases are useful in performingspecific binding assays. In general they are of three types: preformedparticles, the surface of a vessel, and soluble polymers which can beattached to specific binding components and which can be made insolubleduring the binding assay. For each of these solid phase types, thespecific binding activity may be an inherent property or it may begenerated by covalent or noncovalent attachment of materials,hereinafter called "specific binding agents", which confer specificbinding properties on a solid phase.

Preformed particle solid phases include stabilized microbial suspensionssuch as a Staphylococcus aureus strain which naturally produces theimmunoglobulin-binding molecule, "Protein A". Alternatively, the solidphase can be non-microbial particle suspensions of minerals(hydroxyapatite, glass, or metal), beaded insoluble polymers (such asdextran [Sephadex G-10 or G-25], agarose, or polystyrene). Some of thesenon-microbial particles naturally exhibit useful binding activity (e.g.hydroxyapatite). However, most others must be coated with a suitableagent, using coating procedures well known in the art. These solidphases noted above can also be prepared with or may exhibit inherentmagnetic or paramagnetic properties which may be exploited forseparating bound from unbound label or for mixing.

Small particles confer rapid reaction kinetics on solid phase assays,but excessively small particles are not ideal for centrifugalapplications. For most applications, particles should have averagediameters of 0.5-3 microns and densities of 1.1 gm/mL or greater.Preferred particles have relatively uniform diameters of approximately 1micron and densities of 1.5-3.5 gm/mL. The preferred use of micron-sizedmicroparticulate solid phases results in surprisingly fast reactionkinetics, comparable to liquid phase assays.

For gravity separation embodiments, preferred solid phase materialsinclude very high-density particles, such as glass or plastic-coatedmetal beads (typically 3-6 mm diameter). Coated metal beads can easilybe produced by immersing the metal beads in a solution such aspolystyrene dissolved in acetone or chloroform, then draining the beads,allowing the solvent evaporate, then incubating the beads with one ormore specific binding agent such as antibody, as is well known in theart.

Some particles specifically bind analyte with a non-biologicalmechanism. In one such embodiment, glycosylated hemoglobin binds to ionexchange particles from BioRad (Richmond, Calif.), and especially toparticles with boronic acid on their surfaces such as those from PierceChemical Co. (Rockford Ill.). Such particles are used for determiningthe percentage of this analyte in blood using column chromatography, andthese or related particles are suitable for serving as bindingcomponents in the methods of the present invention.

Binding components can also be produced by precoating the assay vessel.The most stable precoated assay vessels will be produced by chemicallycross-linking the molecules which contribute binding activity to eachother and/or to the assay vessel surface. Such coated assay vessels(anti-IgG for mouse, rabbit, goat) are commercially available, forexample, from Micromedic Systems, Inc. (Horsham, Pa.).

Alternatively, the solid phase can be produced during or subsequent toincubation of the assay mixture, by polymerization or aggregation ofsoluble subunits coupled to a useful binding agent. Since reactionsequilibrate more rapidly when all reactants are in solution, such anapproach offers shorter incubation times than traditional methods usinglarge, preformed, insoluble binding components.

In immunoassays, binding components will typically contain specificbinding agents such as antibody, antigen, protein A, avidin, or biotin,either adsorbed or chemically coupled to the solid phase. A preferredsolid phase coating for immunoassays is species-specificanti-immunoglobulin (for example, goat anti-rabbit IgG).Anti-immunoglobulin coated particles can be produced using bacterialparticles (Frohman et al., J. Lab. Clin. Med. 93:614-621, 1979, andBennett and O'Keefe, J. Biol. Chem. 253:561-568, 1980 hereinincorporated by reference). For maximum stability, such preadsorbedbinding components can be chemically stabilized (e.g. withglutaraldehyde or carbodiimide) to cross-link binding agent molecules toeach other and/or to the binding component particle surface. Thesemodified "biological" solid phases have the advantage that they do notexperience interference from immunoglobulin molecules such as occur athigh levels in serum samples, and are commercially available(Tachisorb™, from Behring Diagnostics, La Jolla, Calif.).

Preferred particulate solid phases for centrifugal applications arethose which have appropriate density and particle size to spin downrapidly through primary layer materials, preferrably in standardlaboratory and clinical centrifuges. These include carboxylatedbromostyrene latex particles (JSR Corp, New York, N.Y.) and similarsized carboxylated magnetic copolymer particles (Seragen, Indianapolis,Ind.), and silica particles (3 micron average diameter, Baker ChemicalCompany). For example, these particles can be rapidly pelleted at2000-3000×g (at 45° C.) using primary layers comprised of dibutylphthalate, dimethyl cinnamate, or dimethyl itaconate. Surprisingly, theglass particles will even sediment through such primary layers withoutcentrifugation.

Preferred also are the characteristics of low non-specific binding ofthe label to be used (usually 1-2% or less) and a high, reproduciblymanufacturable binding capacity (typically 10-50 microgram IgG per mL of10% wt/v suspension). Commercial preparations of S. aureus (BehringDiagnostics, San Diego, Calif. and Imre Corp., Seattle, Wash.) exhibitthese desirable properties. Chemically stabilized, anti-immunoglobulincoated S. aureus suspensions with these properties are also availablefrom Behring Diagnostics (Tachisorb).

Other desirable solid phases for embodiments employing centrifugalseparations include Sephadex G10, G15, and G25 (Pharmacia), which can beoxidized with periodate to form aldehydes suitable for chemicallycoupling with amino groups on proteins and other molecules. Becauselarge molecules are excluded from the matrix of these particles,nonspecific binding of most labels is very low and can be furtherminimized by including in the assay solution appropriate chemical agents(such as sodium chloride >0.1M).

D. ASSAY METHODS

For simplicity, the specific binding assays of this invention will bedescribed in terms of antigens and antibodies. However, it will beappreciated by those skilled in the art that any substantially specificbinding pair can be employed in the methods of this invention,including, but not limited to, the following: the binding ofcomplementary nucleic acid sequences; the binding of lectins withcarbohydrates; the binding of hormones with receptors; the binding ofvitamins with transport proteins; and the binding of immunoglobulinswith nonimmunoglobulin, antibody-binding proteins.

The assays of this invention can employ any of a variety of labelingsubstances which are well-known in the art. These can include, but arenot limited to, the following: radioisotopes (eg. 32-P, 3-H, 125-I,35-S, 14-C); enzymes (eg. horseradish peroxidase, urease, betagalactosidase, alkaline phosphatase, glucose oxidase, enteropeptidase);fluorophores (eg. fluorescein, rhodamine, dansyl, phycobiliproteins,Nile blue, Texas red, umbelliferone); luminescers or luminescent sourcematerials; transition metal chelates; enzyme substrates, cofactors, orinhibitors; particles (eg. magnetic, dye, high refractive index); andzymogens. These are exemplified in part by the following publications:U.S. Pat. No. 4,181,636; U.S Pat. No. 4,401,765; U.S. Pat. No.3,646,346; U.S. Pat. No. 4,201,763; U.S. Pat. No. 3,992,631; U.S. Pat.No. 4,160,016, U.S. Pat. application Ser. No. 486016 (EP 0123265A1), allof which are herein incorporated by reference.

The various functional configurations in which specific binding assayscan be performed are well known in the art and are described extensivelyin, for example, Maggio (ed.), Enzyme Immunoassay. CRC Press, BocaRaton, Fla., 1980, herein incorporated by reference. Severalrepresentative examples employing the methods of the present inventionare described below. These methods may be used to detect the presenceand/or amount of a wide variety of analytes. Representative analytes arelisted in EP 123,265.

Briefly, in a competitive immunoassay, sample suspected of containingantigen (analyte) and a known amount of labeled antigen (tracer) competefor a limited amount of analyte-specific antibody. In heterogeneouscompetitive immunoassays, anti-immunoglobulin antibody or Staphylococcalprotein A immobilized on a solid phase to form a binding component isadded at the same time or in a subsequent step. Following incubationduring which specific binding occurs, the binding component is passedthrough the layer(s) of the cushion, thereby separating bound label fromunbound label. In homogeneous competitive enzyme-labeled assays, theassay mixture can pass through the cushion to mix with enzyme substratecolor developer in a secondary layer.

The binding component (in a heterogeneous assay) or the assay mixture(in a homogeneous assay) can pass through the cushion due to gravity orthe assay vessel can be subjected to a centrifugal force. If the bindingcomponent is magnetizable or magnetic, the assay vessel can be subjectedto a magnetic field to move the binding component through the cushion orfor mixing. The presence or amount of bound label is then determined bymeans appropriate to the label, and is related to the presence or amountof analyte initially present in the sample, by comparison to a series ofknown standards. For instance, gamma counters or scintillation countersare appropriate for detecting radioisotopes, spectrophotometers areappropriate for detecting substances or solutions which absorb light,etc.

All the reagents comprising the reagent mixture (including bindingcomponents and label) can be premixed and the assay initiated by theaddition of sample. In this case, the reaction typically will be allowedto substantially or completely equilibrate before the binding componentor assay mixture is caused to pass through the primary layer. In such anembodiment, precise timing of the incubation period is not required.Alternatively, sample and label can be premixed and added simultaneouslyto the reagent mixture and incubated for a fixed interval to form anon-equilibrium assay mixture, then the binding component (forheterogeneous assays) or the entire mixture can be caused to passthrough the primary layer.

A preferred alternative protocol for a competitive immunoassay is topredispense binding components comprising antibody-capture particles, aswell as label, to form a reaction mixture, with analyte-specificantibody isolated in a reagent reservoir in the assay vessel. Antibodycan be delivered to the assay mixture to initiate the binding reactionusing, for example, low speed centrifugation. Very high precision can beexpected where all reagents are factory dispensed, and wheresimultaneous delivery of antibody to all assay vessels in a centrifugeinitiates the reaction, and where the reaction is terminatedsimultaneously in all assay vessels when the binding componentspenetrate the primary layer. When standards and controls are included insuch assays, critical timing and temperature control are not necessary,a run size is limited only by the centrifuge capacity (which can exceed200 for some microcentrifuges).

As an alternative to competitive binding assays, a heterogeneoussandwich assay can be performed. For sandwich immunoassays, analyte isincubated with two antibodies which can be present in excess, one beingimmobilized, or capable of being immobilized (being the bindingcomponent), and the other conjugated to a label. The antibodies can bedirected against two different, non-competing determinants (epitopes) onthe analyte or, if there is a multiply repeated determinant on theanalyte, they can be directed to the same determinant.

Sandwich immunoassays can be carried out in simultaneous, forward, orreverse configurations (as described in U.S. Pat. No. 4,376,110, hereinincorporated by reference), depending upon the order in which theanalyte and the antibodies are added. Labeled antibody which is boundvia analyte to the solid phase is then separated from unbound labeledantibody by passage through the cushion, as described above, and theamount of bound label determined using means appropriate to the label.

Some sandwich assays require addition of binding component, followed byseparation of bound and unbound analyte, then followed by addition oflabel (labelled antibody). In the present invention, the addition oflabel to the binding component could occur in a secondary layer. Thishas the advantage of eliminating a manual user step in such an assaymethod, adding convenience and reducing the opportunity for error.Selective movement of the binding component to a specific secondarylayer prior to its movement to the most distal secondary layer can beachieved using an appropriate sequence of applied forces and selectionof primary and secondary layer materials to have appropriate densities.For example, low speed and high speed centrifugation could be employedto cause the binding component to pass first to an intermediatesecondary layer, then to pass through more distal, denser layers.Alternatively, a water-immiscible secondary layer could be employed witha melting temperature higher than the temperature maintained during thefirst separation step. The temperature could be raised above the meltingpoint of this solid secondary layer in order to complete the assay.

Sandwich assays offer the advantage that both antibodies can be presentin excess, hence the sensitivity of the assay is not strictly limited bythe affinity constant of the antibody(s).

In one special application of the present invention, a noncompetitivesandwich binding assay is used to detect antibody in a sample, and thusis useful in clinical serology and in screening hybridoma cultures. Forexample, either anti-mouse IgG or antigen can be coated on the solidphase as described above. Substantial reduction in manipulations can beachieved using the present invention compared to standard proceduresused in hybridoma screening. An added advantage is that where antibodyis bound to the solid phase, rapid selection of high affinity antibodiesis possible by detecting binding to subnanomolar levels of labelledantigen.

E. ASSAY VESSELS FOR INCUBATIONS AND SEPARATIONS

The vessel in which the cushion (primary and any secondary layers) iscontained is referred to herein as the "assay vessel". The assay vesselmay also contain one or more components of the reaction mixture.Numerous geometric configurations using different sizes and shapes ofassay vessels are possible within the scope of the present invention.Referring now to FIG. 1A, in most applications the cushion, herecomprising a primary layer 12, is contained within an assay vessel 10which is closed at its distal or bottom end 13.

The assay vessel has a substantially cylindrical body 14 which definesan elongated chamber 16. The primary layer 12 extends generallytransversely within the chamber to form a barrier therein, typicallyfilling approximately 1/3 to 7/8, and preferably filling 15/24 to 3/4 ofthe volume of the chamber. The optimal volume of the primary layer willbe determined in part by the geometry of the assay vessel, the nature ofthe label, the detection method and device, if any, and the shield, ifany.

Where both primary and secondary layers are utilized, typically thevolume of the secondary layer will be equal to or greater than thevolume of the primary layer. When more than two layers are used, thedistal layer is typically the largest. It will be evident to one skilledin the art that the ratio of the volumes of primary to secondary layersused will be influenced by the nature of the particular layer materialsused, and the nature of the label and binding components used. Forexample, where an enzyme is used as the label and an enzyme substrate isan additive to a secondary layer, the ratio of primary to secondarylayers will be low (typically as low as 1:10) in order to achievemaximal sensitivity. In contrast, in the case where the label is afluorescent material and a secondary layer is utilized to provide theoptimum solvent environment for detection, the ratio can be high(typically as high as 5:1).

Suitable assay vessels include test tubes and wells, or strings of wellsin a multiwell plate. It is preferred that the assay vessel beresealable at the top or proximal end 17, to protect the user and theenvironment from biohazards or chemical hazards in the sample or assayreagents. It is also preferred to provide the assay vessel with apenetrable septum 18. While a simple metal foil or polyethylene film issufficient for this purpose, a seal with elastic properties such as, forexample, a septum made from rubber (e.g. silicon, neoprene, or EPDM) orfrom a heat-meltable, moldable, rubberlike plastic (e.g. Kraton®thermoplastic rubber from Shell Oil Co.) is preferable.

Even more preferable, for ease of manufacturing plus ease and safety inuse, is a resealable septum which is penetrable by a blunt-endedinstrument, such as a blunt needle or a disposable pipette tip.Particularly preferred is a resealable, elastic septum which has beenmolded with a thin region, or partially or completely precut with aslit, so that air can vent during the addition of liquid assayreactants. Such vessels are essentially permanently sealed at the timeof manufacture, eliminate the handling of caps by the user, yet allowsafe and convenient addition of assay reactants and/or sample by theuser.

For radioisotopic applications, the assay vessel may be composed ofpolyethylene or, more preferably of polypropylene for its strength andsolvent resistance. Non-isotopic methods typically benefit from maximumclarity of the assay vessel, which can be made from glass, polystyrene,polycarbonate, nitrocellulose, and optical grade polypropylene (producedwith clarifying additives from Milliken Chemicals, Spartanburg, S.C.). Asurprising feature of the present invention is that test tubes composedof clear plastic such as polystyrene, which are desirable fornonisotopic assays, can be used with several of the primary layermaterials even though such plastics are known to be vulnerable to damageby organic solvents and hydrocarbon oils. Adhesion of rubber and otherseptum materials to plastic or glass tubes can be readily accomplished.In one embodiment, a tight fitting molded cap is used with an elasticseptum containing a precut slit. In another embodiment, a disk ofrubber, precut with a slit, is fastened permanently to the flange at thetop of a tube using methods well known in the polymer industry. Forexample, silicone adhesive will effectively bond silicone rubber to maykinds of tubes, including glass and some plastics. With appropriatechemical priming, polypropylene tubes can be glued to various rubbers,such as EPDM polymer blends. Cyanoacrylate adhesive will bond EPDMrubbers to polypropylene even without priming.

In one preferred embodiment especially suited for isotopic bindingassays, the assay vessel is a 0.4 milliliter microcentrifuge tube(approximate dimensions 5×45 mm) composed of polypropylene, such as iscommercially available from Sarstedt (Princeton, N.J.), West CoastScientific (Emeryville, Calif.), and from numerous other manufacturersand distributors.

As shown in FIG. 1A, during use of the assay vessel, an assay mixture 24including specific binding components 26, is placed into contact withthe primary layer 12. Substantially concurrent with separation of thebinding components from the unbound label in the assay mixture, thebinding components will enter the primary layer and will typicallycontinue to the distal end 13.

Referring now to FIG. 1B, an alternative larger (2 mL) embodiment of theassay vessel 10 is shown. Other similar embodiments employ test tubeswith external dimensions such as 8 by 55, 10 by 55, 10 by 75, 12 by 55,and 12 by 75 millimeters. Within this embodiment, the chamber 16 definedby the assay vessel is of a size sufficient to receive one or morepreformed beads which are initially positioned on the upper surface ofthe primary layer 12, which is in a solid form. Specific binding agentsare attached to the beads to form binding components 26. As shown inFIG. 1B, the cushion comprises a primary layer 12 and a secondary layer28. The primary layer 12 will be the only layer to contact the assaymixture 24. Following incubation and conversion of the primary layer toa liquid form, the binding components with bound label pass through theprimary layer, enter the secondary layer, and settle to the distal end13 of the assay vessel. As an alternative to the cap 20 shown in FIG.1A, the assay vessel may be provided with a threaded portion 30 which ismateable with a suitable cap (not shown).

In an embodiment related to that shown in FIG. 1B, employing a liquidprimary layer and typically lacking secondary layers, the bindingcomponents 24 are initially positioned at the distal end of the assayvessel, and are then incubated with the other components of an assaymixture. Finally a primary layer material is poured or otherwisedispensed into the assay vessel, leaving the washed binding componentsand bound label at the bottom of the assay vessel, with the othercomponents of the assay mixture (including free label) displaced to thetop of the primary layer. This embodiment is also effective where thedistal inner surface of the assay vessel has been coated to form thebinding components.

Referring now to FIG. 2, another preferred embodiment is shown which issimilar to that shown in FIG. 1B, with the use of a well 32 within amultiwell plate. An alternative embodiment which is preferable for someapplications uses strips of 1 or 1.4 mL tubes (8 millimeter outsidediameter, Skatron A. S., Lier, Norway) which fit into a standard 96 wellplate array. These embodiments can be sealed with penetrable septa, aretypically used with non-isotopic labels, and are appropriate forseparations achieved by centrifugal, gravitational, or magnetic forces.The well 32 generally comprises a body 34 defining an open space 36. Thewell is of a size sufficient to receive one or more preformed beads,plus a predispensed primary layer, and some cases one or morepredispensed secondary layers. In the embodiment shown in FIG. 2, thebeads are initially positioned on the upper surface of the primary layer12. The beads have specific binding agents attached thereto, thusforming binding components 26. Positioned below the primary layer is asecondary layer 28. Following incubation and conversion of the primarylayer to a liquid form, the binding component(s) with bound label passthrough the primary layer, enter the secondary layer, and settle to thebottom of the well. Because of the short distance from the top of theprimary layer to bottom of the well, this embodiment is especiallyappropriate for separations employing magnetic forces.

Shielding is typically not needed in the embodiment shown in FIG. 2because signal generation occurs only in a layer separated from thesecondary components containing free label. A preferred embodimentutilizes an enzyme label, a primary layer which is in a solid formduring incubation and which is converted to a liquid form prior toseparation, and a secondary layer which includes an enzyme substratewhich produces detectable signal in the presence of label.

In another preferred embodiment wherein the label is fluorescent and thedetector "looks" up through the bottom of the well, side-excitation inthe bottom region of the cushion can be used to prevent excitation offree label. Alternatively in such cases, a quenching agent (such as aresonance energy transfer receptor like rhodamine where fluorescein isthe label) can be added to binding assay. The use of fluorescentquenching compounds has been described for homogeneous binding assays(Ullman and Schwarzberg, U.S. Pat. No. 3,996,345, herein incorporated byreference). Such a quencher may be useful in a heterogeneous bindingassay because it will quench fluorescence of unbound label but not thatof bound label, since it will be removed from the binding components bythe cushion. In certain cases where the aqueous compartment of aparticulate solid phase is not removed by passage through a cushion,inclusion in the assay mixture of such a quencher would be particularyuseful to reduce non-specific signal.

F. SHIELDS

Depending upon the nature of the signal emitted or produced by the labeland the height of the cushion, it may or may not be desireable tophysically shield the portion of the vessel containing the secondarycomponents (with free label) in such a way that only signal emitted fromthe binding components is detected. Referring now to FIG. 3, a reusabledetection vessel is shown with an assay vessel place therein. Thedetection vessel 38 generally comprises a body 40 defining an interiorchamber 42. If, for example, the label is a gamma emitting isotope theupper portion (and in some cases the extreme distal end) of thedetection vessel 38 could be provided with a metallic or metallizedshield 44, composed preferably of lead or leaded plastic, or of copper.If the label is a fluorophore or a luminescer, the upper portion of thedetection vessel could be provided with a shield composed of alight-impenetrable material. It will be apparent that in certainapplications, different assay vessels and different shields will bepreferable.

If shielding is desireable, the shield 44 can be either integral in thedetection vessel body or it can be a separate shield, enclosed by thebody of the detection vessel, into which the assay vessel 10 fitsslideably. The latter configuration is generally preferred for itsdurability and superior geometry for shielding. For best shieldingperformance, the bore of the shield will typically be cylindrical and ofthe minimum size required for convenient insertion and removal of theassay vessel.

Referring again to FIG. 3, the assay vessel 10 fits slideably into ashield composed of a radiation-shielding material. The shield is open atboth ends and has an inner diameter which is sufficiently greater thanthe outside diameter of the assay vessel to allow the assay vessel toslide easily into the shield. A particularly convenient configuration isone in which the assay vessel is a test tube which has a lip whichengages the top of the shield and supports the tube within the shield.Microcentrifuge tubes having an approximate volume of 0.4 mL arecommercially available from a number of sources and will slide easilyinto and out of a shield of inner diameter approximately 1/8 inch indiameter. Tubes with a similar outside diameter, but which are longerthan 0.4 mL tubes, would be advantageous in certain applications.

Because in this embodiment the assay vessel is small in diameter, theshield can also be small in diameter; hence, there is comparativelylittle scattered radiation detected from the supernatant or the cushion.Therefore the detection of bound radioisotopic label is essentiallyunimpeded by the inadvertent simultaneous detection of unbound label,unlike with prior art devices and methods.

The composition of the shield will vary, depending upon the nature ofthe signal emitted or produced by the label, but its design and materialwill typically be sufficient to block detection of at least 90%, andmore typically greater than 99%, and optimally greater than 99.7% of thelabel in the unbound fraction after separation of bound (solid) andunbound (supernatant) components of the assay mixture.

For example, if the label is a gamma-emitting isotope such as125-Iodine, the shield might be composed of lead, leaded plastic,copper, or other suitable material. For detection instruments comprisinggamma counters with annular crystals (including Micromedic Systems,Horsham, Pa., LKB Instruments, Gaithersburg, Md., and BeckmanInstruments, Brea, Calif.), a sleeve of 1/8 inch thick lead (3/8 inchO.D., 1/4 inch I.D.) provides an excellent combination of strength (towithstand manufacturing manipulations and centrifugation at least up to3000×g in use) and radiation shielding. However, MacKenzie (J.Immunological Methods. 67:201-203, 1984, herein incorporated byreference) has calculated that a much thinner (1 mm) sheet of leadblocks 99.999964% of a dose of 1 25-Iodine. Thus to achieve 99.0%shielding theoretically requires a lead foil only 36 millionths of aninch thick.

High-integrity lead foils (0.006 inch and 0.012 inch thick) arecommercially available (Nuclear Associates, Carle Place, N.Y.) andprovide essentially complete radiation shielding with much less weightthan a 1/8 inch thick sleeve. Lead foil could be used to form a shieldin applications where the 1/8 inch thick sleeve is undesirably heavy.Lead-coated or lead-containing composite plastics or fabrics, producedfrom molded lead or lead foil are also effective lightweight shieldmaterials. For such foils and thin films, strength is provided by aplastic support sleeve. Other materials including non-lead metals suchas brass can be used as shields for radiation including that emitted by125-Iodine.

If the label is a beta-emitting isotope such as tritium or32-Phosphorus, the shield might be composed of an opaque plastic. If thelabel is a fluorophore or a luminescer, the shield might be blackplastic. However, in most applications, labels such as fluorophores andlow energy beta-emitting radioisotopes will not require shields.

Where required, the shield is designed to mask approximately the upperseventy-five percent of the assay vessel and usually not more thanapproximately the upper ninety percent of the assay vessel. A generalpurpose shield will typically be as long as possible withoutsignificantly reducing the detectable label in the unshielded portion46. For gamma counters with annular crystals and assay vessels such asshown in FIG. 1A, a 3/8 inch O.D, 1/8 inch I.D. lead sleeveapproximately 13/8 inch long is preferred. Such an assay vesseltypically contains approximately 250 microliter of cushion liquid and 10microliters or less of binding components. However, for certaindetection instruments and for different cushion heights, modificationsin shield length or in the volume of cushion and/or binding componentswill be desirable.

In cases where the detector is centered near the bottom tip of the assaytube, part or all of the assay mixture may not need to be shieldedlaterally because the shield below will block undesirable radiation.This form of shield is effectively a skirt, and has the added advantagethat the assay mixture can be directly observed (as during reagentadditions to the top of the cushion) even while shielding is in effectand the assay vessel is in its final position.

In addition to accommodating the assay vessel within itself, the shieldshould fit inside the body of the detection vessel, as shown in FIG. 3.The detection vessel is closed at the bottom and may or may not besealable at the top as well. Typically, the body of the detection vesselis a test tube, the inner diameter of which is sufficiently greater thanthe outside diameter of the shield to allow the shield to slide tightlythereinto for purposes of semi-permanent assembly. As shown in FIG. 3,the shield may be provided with a shim 48, preferably composed of anadhesive paper label, glue, or a suitable resilient material, in orderto maintain the position of the shield within the detection vessel.

Suitable for use as detection vessel bodies are test tubes ofpolypropylene, polyethylene, or glass, typically having approximateouter dimensions 12×75 or 12×55 mm. Such tubes are commerciallyavailable from a variety of sources and are advantageous in that theyfit readily into gamma counters and/or scintillation counters. Where 0.4mL assay vessels have tethered caps which bind on the inner walls of thedetection vessel, a simple tool (e.g. Model 61-008 from the Stanley ToolCompany) can be used to insert and withdraw assay vessels.Alternatively, a shorter (12×55 cm) detection vessel can be used withsuch assay vessels because assay vessels with tethered caps can beeasily inserted and removed without a tool.

In general, plastic tubes (especially polypropylene) are preferred overglass tubes for use as detection vessel bodies because there is lessrisk of breakage and they can typically withstand greater centrifugalforce. In general, too, it is preferred that the detection vessels bereuseable.

In a preferred embodiment for centrifugation of assay vessels directlyin shields made with the 3/8 inch outside diameter lead cylindersdescribed above, the detection vessel would contain the shield therein,supported by a cylindrical member 50. Such a cylindrical member 50 ispreferably composed of plastic such as polystyrene, and may be closed ata level distal from the shield so as to support assay vessels at aconstant height.

While most commercially available gamma counters exhibit good shieldingusing the 3/8 inch outside diameter lead cylinder described above, somewith well-type crystals (especially many gamma counters having more thanfour crystals) require a modification in the shield design. The supportcylinder closed at one end as described above can contain a shieldingdisk 52, made from a suitable shielding material such as lead. This diskis positioned at the bottom of the well formed by the member 50 toshield the gamma counter from unbound label radiation which is travelinggenerally parallel to the long axis of the assay vessel. A surprisingadvantage of this design is that improved shielding is obtained with allgamma counters, while causing only a slight decrease in detectability ofthe bound label located in the distal end of the assay vessel.

Configured in the manner described above, namely, an inner assay vesselprefilled with a cushion as described herein and slideably fitted into ashield within the body of a detection vessel, where the shield issupported by a cylindrical member, a specific binding assay can berapidly and conveniently performed in a self-contained microtube with aslittle as one liquid addition (sample) step and one brief centrifugationstep prior to detection of bound label. In certain instances, thecentrifugation step can be eliminated. For example, one such situationis where gravity separation is employed using dense particles and ameltable primary layer. These binding assay methods can use equipmentcurrently available in most laboratories which perform such assays.These assays can be accomplished with considerable reduction in time,skilled labor, and radioactive waste volume over specific binding assaymethods as currently practiced. Comparable advantages will beexperienced for both isotopic and non-isotopic applications.

G. COMBINED USE OF CUSHIONS AND SHIELDS

Surprising and valuable features are inherent in the combined use ofwater-immiscible primary layers and radiation shields. Even the mostconvenient currently available isotopic assays using antibody-coatedtubes or large, antibody-coated beads must be processed both before andafter incubation by skilled persons or by sophisticated liquid handlingequipment. Such processing includes post-incubation addition of washsolution, aspiration or decanting to remove free label, and usually arepeat of these steps. Not only are these steps inconvenient, they riskspills and contamination from both biohazards in the sample andradioactivity from the components of radioisotopic assays.

Unless carefully controlled, this washing can be disadvantageous forseveral reasons. First, assay precision and accuracy can suffer fromdissociation of antibody-antigen complexes which occurs during thewashing step, potentially reducing signal. This is especiallysignificant with monoepitopic assays (such as with small antigens or inmany assays using monoclonal antibodies), where a single attachmentbetween antibody and antigen is formed. Furthermore, the wash liquidvolume in conventional heterogeneous binding assays must besignificantly larger than the volume of the assay mixture, and thelarger the wash volume, the more effective the washing procedure. Whenthis wash solution is removed, typically by decanting onto an adsorbentpad, a significant increase in radioactive waste volume is producedcompared to the initial mixture volume.

A valuable and surprising feature of the present invention is that theabove described wash solution can be eliminated and the reagents can bekept totally contained in the assay vessel. This feature providesimproved safety compared to conventional methods because potentiallyhazardous materials (for example, radioactivity and/or infectiousmaterial) is totally contained for safe and convenient disposal.Subsequent to loading the assay mixture, the need for special skills orcare is eliminated. Another surprising feature is that thewater-immiscible layer can be small relative to the volume of themixture mixture, and much smaller than the typical wash volume used intraditional heterogeneous assays. As the binding components pass throughthe cushion, they continuously encounter fresh cushion medium and thusare effectively washed in a small volume.

The above configuration also represents a significant improvement overprior art shielding methods because the introduction of an immisciblephase between the assay mixture and the binding component dramaticallyincreases the preciseness and completeness of separation of the boundfrom unbound label fractions. This immiscible phase coupled with theshielding features described above allow one to effectively performself-contained binding assays such as radioimmunoassays. Separation ofbound and unbound label in such assays is virtually instantaneous andcan produce equilibrium binding assay data for applications incharacterizing the tightness of interaction for binding pairs.

The geometry of the assay vessel and shield, both being elongated andrelatively small in diameter, virtually eliminates the contribution ofscattered radiation to the total signal measured, hence practically nomathematical correction of the data is required. Because the assaymixture and its components are immiscible in the primary layer, neitherdilution nor dissociation occur during incubation of the mixture mixturein contact with this layer, and no dissociation of binding pairs occursas is observed in the prior art using sucrose and related materials asbarriers. Thus the entire assay including mixing and incubation stepscan occur in contact with the primary layer, eliminating the need totransfer the incubated mixture mixture onto a cushion, or tocontrollably inject a washing solution of material such as sucrose underthe incubated mixture mixture, as in the prior art.

A further feature of the present invention is evident with the use ofbinding components attached to the assay vessel or to large, densebeads. A water-immiscible cushion denser than the secondary componentsbut less dense than the large beads (if any) can be added at the end ofthe assay if desired, achieving separation of bound from free labelwithout requiring the removal of unbound label and other assay mixturecomponents from the assay vessel.

A further attribute of binding assays employing incubation of an assaymixture on a water-immiscible liquid is the dramatic reduction in thevolume of the mixture. Manipulation of a visible pellet is not requiredand the assay mixture components can be predispensed onto the top of theprimary layer. Such predispensed assay mixture component can be storedas liquids, or concentrated and/or stabilized by lyophilization, thenrehydrated or diluted for use by the addition of a small (e.g. 10microliters) sample.

Thus the assay can be miniaturized, waste dramatically reduced, andsafety significantly increased, while simultaneously saving labor andreducing error-producing steps in the performance of specific bindingassays.

H. UNSHIELDED CUSHION EMBODIMENTS

For unshielded applications, especially using enzyme or fluorescentlabels in multiwell plates, the use of an immiscible primary layer andan aqueous secondary layer makes possible effective separation ofbinding components from free aqueous label (by gravity, centrifugation,or magnetic forces) over a distance too small to be effective withwholly aqueous cushions. Especially useful in such applications areprimary layers which are readily solidified by cooling, or which aresolid at storage and/or incubation temperatures in the range of 15°-50°C., and can be liquified (typically melted) for the separation step inthis temperature range. Very dense binding component solid phaseparticles (e.g. glass or metal spheres) can be used which will sinkthrough the primary layer when it is liquified by warming. It will beapparent that methods using the present invention are compatible withexisting automated clinical analyzers designed for colorimetric andfluorometric clinical assays.

The following examples are offered by way of illustration, not by way oflimitation.

EXAMPLES

Abbreviations used in the examples include PBS (phosphate bufferedsaline), BSA (bovine serum albumin), TGF-α (transforming growth factoralpha), hEGF (human epidermal growth factor), RIA (radioimmunoassay),DTT (dithiothreitol), and CPM (counts per minute).

EXAMPLE I MINIATURIZED COMPETITIVE RADIOIMMUNOASSAYS FOR TRANSFORMINGGROWTH FACTOR ALPHA

All assays employed as assay vessels 0.4 mL microcentrifuge tubescontaining 0.25-0.3 mL of cushion material. Labeled peptide was producedby chloramine T iodination (specific activity ranged from 200-500μCi/μg). Unless otherwise specified, all solid phases were preparedusing S. aureus suspensions. Centrifugations were for 30-60 seconds in amicrocentrifuge at approximately 10,000×g.

(A) RIA Using Peptide fragment and Butyl Phthalate Cushion:

The synthetic peptide used for immunization of rabbits was a protein andglutaraldehyde conjugate of the c-terminus 17 amino acids of rat rTGF-α(Marquardt et al., Science 233:1079-1082, 1984). This peptide(unconjugated) was also used as a reference standard and as the label(125-iodine labeled). Antiserum or normal rabbit serum (for nonspecificbinding determinations) was adsorbed onto a commercial preparation offormalin-fixed S. aureus (Imre Corp, Seattle, Wash.) to form an antibodysolid phase suspension with 5% solids in PBS. Label cocktail wasprepared by mixing, in 500 μL total volume, 100 μL (250,000 CPM) oflabeled peptide, 100 μL of 10% (0.65M) dithiothreitol, 30 μL of 10 mg/mLBSA, 5 μL of 10% sodium azide, 50 μL of 10x PBS, and 215 μL distilledwater.

Into each assay vessel was loaded 30 μL of label cocktail, 40 μL ofsample, and 30 μL of antibody suspension. After mixing the assay mixtureand incubating overnight at 4° C., the assay vessels were centrifuged,then placed in radiation shields (FIG. 3) and counted in a BeckmanLS-200C scintillation counter using Gammavials (Koch-Light Ltd, Suffolk,England; counting efficiency was ca. 40%).

The data obtained using synthetic peptide calibrator is shown below.Bioactive synthetic rat and human TGF-alpha gave competition curvesequivalent to peptide fragment on a molar basis, with 50% competition atapproximately 0.6 nM peptide.

    ______________________________________                                        TGF-α RIA: PEPTIDE FRAGMENT,                                            BUTYL PHTHALATE CUSHIONS                                                                                      LABEL                                         SAMPLE*  ANTIBODY               BOUND                                         ______________________________________                                        BUFFER   ANTI-PEPTIDE FRAGMENT   43%                                          0.5 nM   "                      30                                            1.0 nM   "                      24                                            2.0 nM   "                      16                                            10 nM    "                       7                                            BUFFER   NORMAL RABBIT SERUM     3                                            BUFFER   MINUS ANTIBODY SUSPENSION                                                                            0.1                                           ______________________________________                                         *final concentration in assay                                            

(B) RIA using anti-fragment antisera and bioactive synthetic peptide astracer and reference standard, with methyl cinnamate cushions and assayreactants predispensed.

Trans-methyl cinnamate (Table 1, item 11, Aldrich Chemical Co., St.Louis, Mo.) was melted by brief heating in a microwave oven just priorto dispensing into assay vessels. The cushion solidified spontaneouslyat room temperature. The solid phase was prepared as in (A) above indouble strength assay buffer (including 4% NP-40 nonionic detergent).This suspension was stable at 4° C. for at least one year. Labelcocktail (1.5 mL) was prepared using 0.3 mL 10x assay buffer (minusnonionic detergent), 0.6 mL 10% NP-40, 0.58 mL distilled water, and 30μL label concentrate (600,000 CPM) prepared from bioactive synthetic ratTGF-alpha (res. 1-50).

Into each assay was loaded 50 μL of reference standard sample(bioactive, synthetic rat TGF-alpha, res. 1-50), 25 μL of labelcocktail, followed by 25 μL of solid phase suspension. Where indicated,all assay reactants were predispensed and equilibrated for at least 3days at 4° C. prior to initiation of the assay by sample addition andmixing. After the indicated incubation periods, assay vessels werecentrifuged and counted using radiation shields (FIG. 3, lacking disk52) in an gamma counter (Abbott Model 200)

Two temperature and mixing treatments were compared with four incubationtimes. One treatment consisted of incubating at 32° C., just below themelting temperature of the cushion, and mixing at 15 minute intervals.The second treatment consisted of incubating at 40° C., above themelting temperature of the cushion, with mixing only at initiation ofincubation (prior to warming). Both assays yielded low nonspecificbinding, high specific binding, and high competition with referencestandards, even after only 30 minutes of incubation. Detailed resultsare shown below.

    ______________________________________                                        TGF-α RIA: BIOACTIVE PEPTIDES                                           & METHYL CINNAMATE CUSHIONS                                                                       TEMPERATURE                                               INCUBATION TIME     32° C.                                                                         40° C.                                     ______________________________________                                        30 MINUTES                                                                    Nonspecific binding 1.1%    1.1%                                              Total bound         24.5    26.5                                              % bound with 0.3 nM sample                                                                        21.9    23.8                                              % bound with 10 nM sample                                                                         5.6     6.9                                               60 MINUTES                                                                    Nonspecific binding 1.1%    1.0%                                              Total bound         28.9    32.2                                              % bound with 0.3 nM sample                                                                        25.2    29.4                                              % bound with 10 nM sample                                                                         6.2     7.1                                               90 MINUTES                                                                    Nonspecific binding 1.1%    1.2%                                              Total bound         28.7    34.1                                              % bound with 0.3 nM sample                                                                        26.2    32.9                                              % bound with 10 nM sample                                                                         6.4     7.6                                               120 MINUTES                                                                   Nonspecific binding 1.0%    1.1%                                              Total bound         31.0    36.4                                              % bound with 0.3 nM sample                                                                        25.6    35.8                                              % bound with 10 nM sample                                                                         6.1     7.6                                               ______________________________________                                    

    ______________________________________                                        TGF-α RIA: PREDISPENSED REACTANTS                                       & METH. CINNAMATE CUSHIONS                                                    Incubation = 120 minutes,                                                     Temperature = 37° C., Total CPM = 7872                                                                   % OF MAX.                                   REACTANTS    LABEL BOUND  C.V.*   BOUND                                       ______________________________________                                        Buffer sample,                                                                               1.6%       18%     N.A.                                        Nonspecific binding                                                                        (146 CPM)                                                        Buffer sample,                                                                             16.8         6%      100%                                        Specific binding                                                                           (1321 CPM)                                                       2.5 nM sample                                                                               8.2         4%      48.6%                                       competition  (642 CPM)                                                        ______________________________________                                         *Coefficient of variation (std deviation/average), N = 6                 

(C) TGF-α RIA using human serum samples with anti-rabbit IgG-coatedsolid phase.

The assay was performed using butyl phthalate cushions as described in(A) above except that the antibody-coated solid phase was preparedeither with fixed S. aureus (Pansorbin™, Behring Diagnostics, La Jolla,Calif.) or with glutaraldehyde cross linked, anti-rabbit IgG-coated S.aureus (Tachisorb™, Behring Diagnostics). The concentration of solids ineach case was the equivalent of 12.5 μL of a 10% w/v suspension per 100μL assay mixture volume. All tubes were preparaed in duplicate andincubated for two hours at 37° C. A 50 μL sample of diluted normal humanserum was added to each tube containing a predispensed cushion, followedimmediately by 25 μL of labelled peptide and 25 μL of antibody solidphase suspension to initiate the reaction.

The results indicate that the even the highest concentration of humanserum had no signficant effect on the Tachisorb assay, while withPansorbin even the most dilute human serum sample caused 41% nonspecificcompetition, presumably by displacing rabbit antibodies bound to proteinA on the solid phase. With Tachisorb, non-specific binding was lower,specific binding was greater, and competion with 1 25 nM standard wasgreater than with Pansorbin. Detailed results are shown in Table 3below.

                                      TABLE 3                                     __________________________________________________________________________    COMPARISON OF PANSORBIN AND TACHISORB                                         ASSAY         LABEL BOUND AS                                                                             BOUND AS % OF                                      CONDITIONS    % OF TOTAL ADDED*                                                                          MAXIMUM BOUND                                      __________________________________________________________________________    Pansorbin, Normal Rabbit                                                                    1.9          N.A.                                               serum, buffer sample                                                          Pansorbin, Rabbit anti-                                                                     27.9         100                                                serum, buffer sample                                                          Pansorbin, Rabbit anti-                                                                     16.4         58.9                                               serum, 1.25 nM Std in buffer                                                  Pansorbin, Rabbit anti-                                                                     14.3         51.3                                               serum, normal human serum                                                     sample (1:10 with buffer)                                                     Pansorbin, Rabbit anti-                                                                     18.1         64.9                                               serum, normal human serum                                                     sample (1:50 with buffer)                                                     Pansorbin, Rabbit anti-                                                                     16.5         59.4                                               serum, normal human serum                                                     sample (1:100 with buffer)                                                    Tachisorb, Normal Rabbit                                                                    1.4          N.A.                                               serum, buffer sample                                                          Tachisorb, Rabbit anti-                                                                     31.7         100                                                serum, buffer sample                                                          Tachisorb, Rabbit anti-                                                                     18.2         57.4                                               serum, 1.25 nM Std in buffer                                                  Tachisorb, Rabbit anti-                                                                     31.2         98.4                                               serum, normal human serum                                                     sample (1:10 with buffer)                                                     Tachisorb, Rabbit anti-                                                                     31.6         99.6                                               serum, normal human serum                                                     sample (1:50 with buffer)                                                     Tachisorb, Rabbit anti-                                                                     30.8         98.5                                               serum, normal human serum                                                     sample (1:100 with buffer)                                                    __________________________________________________________________________     *Total CPM added = 8080                                                  

(D) RIA for human TGF-α using antisera recognizing the complete,bioactive synthetic hormone

Synthesis of hTGF-α (1-50) peptide and immunization:

The sequence of human TGF-α as determined by DeRynck et al. (Cell38:287-297, 1985) was used to synthesize the low molecular weight formof the hormone (residues 1-50) using an automated instrument(Biosearch). The resultant peptide was used to immunize rabbitsrepeatedly using 0.5 mg of peptide at multiple sites.

Immunoassay procedure:

The assay used reference standards and radio-iodinated tracer preparedfrom purified, bioactive synthetic rat TGF-alpha (PeninsulaLaboratories, Belmont, Calif.). Label cocktail was prepared by mixing,in 1.5 mL total volume, 300 μL 10x buffer (0.5M Hepes, 2 mg/mL BSA, 0.2%sodium azide), 600 μL 10% nonidet P-40 (Shell Oil Co.), 580 μL distilledwater, and 30 μL of labeled peptide (rTGF-α, 800,000 CPM). The antibodysuspension was prepared essentially as described in (A) above. To each0.4 mL tube containg 0.25-0.3 mL cushions of butyl phthalate was added25 μL of label cocktail, 50 μL of sample, and 25 μL of antibodysuspension. Where indicated, 10 μL of 1M DTT (freshly dissolved in 0.5Msodium bicarbonate) was added to each assay mixture. After mixing, thetubes were incubated overnight at 4° C., then processed as described in(A) except that the detection instrument was a gamma counter (AbbottModel 200).

The assay detected rat and human synthetic TGF-α (res. 1-50)equivalently, whether or not the peptides were unfolded by reductionwith DTT. Further, the assay detected authentic biological human TGF-αfrom cell culture media conditioned by A375 cells (Marquardt et al.,PNAS 80:4684-4688, 1983). Detailed results are shown below:

    __________________________________________________________________________    PERCENTAGE OF MAXIMAL BINDING WITH COMPETITION FROM SYNTHETIC                 TGF-ALPHA, CORRECTED FOR NONSPECIFIC BINDING                                  UNREDUCED                  REDUCED WITH DTT                                   CONC. RTGF,  RTGF,  HTGF   RTGF   RTGF   HTGF                                 IN ASSAY                                                                            BIOACTIVE                                                                            INACTIVE                                                                             INACTIVE                                                                             BIOACTIVE                                                                            INACTIVE                                                                             INACTIVE)                            __________________________________________________________________________    0.15 nM                                                                             83.2   85.2   82.0   93.8   92.2   93.2                                 0.32 nM                                                                             77.3   76.9   76.9   87.1   87.7   89.3                                 0.62 nM                                                                             68.5   68.9   70.0   79.9   82.0   83.2                                 1.25 nM                                                                             58.5   58.9   64.4   74.4   76.0   78.0                                 2.50 nM                                                                             54.0   48.2   54.7   64.9   70.7   68.6                                 5.00 nM                                                                             45.7   40.0   40.6   47.3   55.9   59.9                                  1.25 nM*           89.7                 63.3                                 __________________________________________________________________________     *BIOLOGICAL TGFALPHA, PARTIALLY PURIFIED FROM CULTURE FLUIDS (A375 CELLS)

EXAMPLE II

ENZYME-LABELLED QUALITATIVE CENTRIFUGAL COMPETITIVE BINDING ASSAY IN 0.4ML TUBES TO DETECT RABBIT IGG IN A SAMPLE USING ENZYME-LABELLED RABBITIMMUNOGLOBULIN AND CUSHIONS CONTAINNG ENZYME SUBSTRATE IN THE BOTTOMLAYER

(A) Reagents: Labelled antibody was affinity purified rabbit anti-goatimmunoglobulin coupled to horseradish peroxidase (Zymed), diluted 1:3000in phosphate buffered saline containing 1 mg/ml bovine serum albumin.The solid phase was a 10% suspension of heat-killed, formalin-fixed S.aureus (Imre Corp, Seattle, Wash.).

The sorbitol substrate cushion solution was prepared by dissolving 22grams of sorbitol in 50 mLs of distilled water, then dissolving 100 mgof chromogenic substrate (OPD, from Zymed, So. San Francisco, Calif.) inone mL of water and adding 0.1 mL of the OPD stock solution and 0.1 mLof 3% hydrogen peroxide to 9.8 mLs of the sorbitol.

(B) Assay: The assay vessels (0.4 mL polyethylene microcentrifuge tubes,West Coast Scientific, Emeryville, Calif.) were then loaded with 0.1 mLof the sorbitol substrate solution, then overlaid with 0.2 mL of dibutylphthalate. Another set of assay vessels was loaded with 0.3 mL ofsorbitol substrate solution.

On top of the butyl phthalate cushion was pipetted 0.05 mL of 10%pansorbin in phosphate buffered saline containing 0.1% sodium azide. Toone tube was added 0.005 mL of rabbit serum, then 0.05 mL of rabbitanti-goat IgG, affinity purified and labelled with horseradishperoxidase (RAG-HRP from Zymed, diluted 1:3000 in PBS containing 1 mg/mLBSA). To the other tube was added 0.005 mL dilution buffer and 0.05 mLof RAG-HRP. After two minutes, tubes were spun for one minute in ahigh-speed microcentrifuge (Fisher model 235B) and examined for signaldevelopment.

The control pellet was immediately "negative" (dark-brown or black) onits upper surface, while the side contacting the tube remained lightamber. The pellet treated with sample was "positive" (light amber incolor). No color developed in the sample layer or in the separate,clearly visible primary cushion layer, where substrate was absent.Surprisingly, only a little color developed in the lower substratesolution, but as expected the sample tube was nonetheless visiblypositive (light yellow) compared to the control tube (amber). Theunexpected concentration of the substrate on the surface of the solidphase itself provided a dramatic concentrating effect, amplifying thedifference between positive and negative samples. While differences inthe substrate solutions were apparent with careful visual examination,the pellets were easily distinguished at a glance. No further changeswere seen over the next 30 minutes while the samples were kept at 25°C., but over the next 2 hours the almost black control pellet becamesomewhat lighter (dark brown), while the light amber sample pelletbecame somewhat darker (light brown or orange in color). No obviousfurther changes occurred, and the two pellets were easily distinguishedafter more than one week storage at room temperature (18°-25° C.). Afterextended storage, the butyl phthalate layer became amber, as ifextracting the chromophore from the aqueous lower phase. The oil layerin the control tube was darker amber, distinguishable by eye from theoil layer from the sample tube.

An analogous experiment using a sorbitol substrate cushion without theintervening oil layer, and using an air space between the sample and thesubstrate cushion also gave visually distinguishable results. Aftercentrifugation, no demarkation of the sample and cushion layers wasvisible. Washing of the solid phase was not as effective since a coloredstreak traced the path of the solids down the wall of the assay vessel.However, the control vessel streak, and pellet, were clearly darkeramber than those in the sample vessel. With time, the entire solution(sample and cushion) became amber, though after one week the controlvessel was overall still darker amber than the sample vessel.

EXAMPLE III MINIATURIZED IMMUNOASSAY USING REAGENTS LYOPHILIZED ONTO TOPOF PRIMARY CUSHION

The reactants are prepared as in example one, except that the sample isomitted and the oil is methyl cinnamate, which is a solid below 36° C.The assay vessels are frozen and subjected to lyophilization in a SpeedVac™ (Savant) under low speed centrifugation. When the reactants aredry, tubes are stored at room temperature. When sample is added (0.05mL), the reactants are rehydrated, and after two hours at roomtemperature, the tubes are warmed to 37°-40° C. and spun as in example 1above and signal measured.

EXAMPLE IV DETECTION OF 32-P LABELLED DNA BOUND TO HYDROXYAPATITE BYCENTRIFUGATION THROUGH A DIBUTYL PHTHALATE CUSHION CONTAININGSCINTILLATION FLUORS

(A) Reagents: ³² P-labelled double-stranded DNA was divided into twoaliquots. One part was boiled for ten minutes, then placed on ice. Eachaliquot (20 microliters, in 10 mM Tris buffer, pH 8.2) received 100microliters of a 10% hydroxyapatite suspension in the same buffer.Cushions were prepared in 0.4 mL microcentrifuge tubes by pipetting 0.3mL of one of the following solutions: (1) butyl phthalate containing 40gm/L omnifluor (New England Nuclear),(2) butyl phthalate containing 1.25gm/L omnifluor, (3) butyl phthalate alone.

(B) Binding assay: Onto each cushion was pipetted 10 microliters of thesuspension containing unheated labelled DNA and solid phase. Thismixture was spun one minute in a Fisher microcentrifuge (model 235B).Tubes were counted using a Beckman LS-100C liquid scintillation counter.

    ______________________________________                                                          CPM,                                                                          EACH CHANNEL                                                CUSHION             32-P    14-C                                              ______________________________________                                        BUTYL PHTHALATE (BPH)                                                                               85    67025                                             BPH + 12.5 mg/L omnifluor                                                                         23035   78835                                             BPH + 40.0 mg/L omnifluor                                                                         47095   102765                                            ______________________________________                                    

When counted using the 14-C channel, the 32-P was detected with orwithout fluor in the cushion. Counting on the 14-C channel in thepresence of a 1/8 inch thick lead shield resulted in less than 10%reduction in counts, indicating that most of the DNA was bound to thesolid phase under these low-salt conditions. These results indicatethat, as tested here, the use of fluor-containing butyl phthalateeliminated the need for a shield, since using the 32-P channel, freelabel which had not entered the cushion would not be detected. Thesedata also show that even on the 14-C channel, which gave somewhat highersignal than the 32-P channel, the inclusion of fluor in the cushion gavemore than 50% greater signal compared to cushions lacking fluor. On thischannel, however, a shield is required to mask the free label in thesupernatant.

Even using the 32-P channel, the background signal caused by radiationfrom supernatant entering the cushion can be greatly reduced oreliminated by using a shield, and that the shielding is more effectivethan when using the 14-C channel. This is demonstrated using the heatedDNA, which bound less completely to the solid phase in this series ofexperiments. The heated DNA was processed on butyl phthalate cushions asdescribed above.

    ______________________________________                                                       CPM, EACH CHANNEL                                              CUSHION          32-P        14-C                                             ______________________________________                                        BUTYL PHTHALATE (BPH)                                                                          110,    170     101355,                                                                             108850                                 BPH + shield                     79860,                                                                              87160                                  BPH + 40.0 mg/L omnifluor                                                                      38255,  41305   132310,                                                                             141855                                 BPH + omnifluor + shield 25825   108995,                                                                             117705                                 ______________________________________                                    

On the 32-P channel, the lead shield with fluor-containing cushion gavealmost 40% less signal the same cushion without the lead shield,indicating significant signal originating from the supernatant or upperportion of the cushion. Approximately 20% shielding of signal wasobtained using the 14-C channel for the same samples.

EXAMPLE V USE OF ANTIBODY-COATED TUBES WITH DISPLACEMENT OF FREE LABELBY ADDITION OF WATER-IMMISCIBLE "CUSHION"

Fifty microliters of either a BSA solution (1 mg/mL in PBS) or antibodyagainst the rTGF-alpha c-terminal 17 residue fragment (prediluted 1:1000in the same BSA solution) was added to 8×50 mm polypropylene tubesprecoated with goat anti-rabbit IgG (Micromedic, Horsham, Pa.). To eachof these tubes was added 50 microliters of 125-iodine labeled peptidefragment (1 nM in PBS with 0.2 mg/mL BSA). After 5 minutes at roomtemperature, duplicate tubes received one milliliter of either dibutylphthalate or a fluorocarbon oil, FC40 (both from Sigma Chemical Co., St.Louis).

The dense oils displaced the aqueous assay mixtures from the bottoms ofthe tubes. Those with dibutyl phthalate required some agitation todislodge droplets of aqueous assay mixtures trapped near the bottom, anda thin film of water appeared to persist between the oil and the tubeinner surface. With FC40, the water floated immediately to the surface,without any apparent retention in the oil phase.

All tubes were counted immediately in a scintillation counter, using13×50 mm plastic tubes as holders for gammavials (Koch-Light), afterwrapping the supernatant and most of the oil layer in a 1.25 inch longcylinder of 0.006 inch lead foil which was supported 7/8 inch above thebottom by a plastic cylinder.

    ______________________________________                                        RESULTS                                                                       assay  primary layer       % bound                                                                              % bound                                     mixture                                                                              material    cpm     (total)                                                                              (specific)                                                                           S/N*                                 ______________________________________                                        antibody                                                                             butyl phthalate                                                                           14680   68.2   49.4   3.6                                  control            3580    18.8                                               antibody                                                                             fluorocarbon oil                                                                          1430    8.3    5.8    3.3                                  control                                                                              (FC-40)      400    2.5                                                ______________________________________                                         *S/N = signal to noise ratio                                             

While both oils produced significant signal, they differed inperformance. Butyl phthalate required some manipulation and yielded ahigh background, but quite high signal considering the short incubationand the relatively high antibody dilution (equilibrium binding at 1:2000antibody dilution would be expected to yield approximately 35-40%specific binding). FC40 yielded a very low background, and a signalcloser to the expected value for a 5 minute incubation. In both cases,the signal to noise ratio was similar.

EXAMPLE VI URINE SAMPLES FROM CANCER PATIENTS TESTED WITH TGF-ALPHA(ANTI-FRAGMENT) AND HEGF RIAS

For the TGF assay, 2.5 mL of urine was desalted through a G15 Sephadexcolumn (PD-10, Pharmacia) which had been equilibrated with ammoniumbicarbonate buffer. The void volume fractions containing urine peptideswere lyophilized and reconstituted with 120 μL of water plus 12 μL of areducing solution containing 1M dithiothreitol and 0.5M sodiumbicarbonate. Myeloma samples received an extra 10 μL of reducingsolution and only 110 μL of water. For the hEGF assay, urine was dilutedfive fold with buffer.

A 0.050 μ sample of each processed urine sample was mixed with 0.025 mLantibody suspension and 0.025 mL of radioiodinated tracer (full lengthTGF-α, residues 1-50, or hEGF, residues 1-53, 250-275 μCi/μG,approximately 10,000 cpm) in incubation/separation vessels containing0.25 mL dibutyl phthalate. After incubating overnight at 4° C., vesselswere centrifuged for 30 seconds at approximately 10,000×g and wereplaced into radiation shields (42 in FIG. 3) and were counted one minutein an LKB Rackgamma counter. Standards consisted full length TGF-α andhEGF in buffer containing 0.2 mg/mL bovine serum albumin and treated inthe same manner as urine samples.

    __________________________________________________________________________    SUMMARY OF TGF/EGF RESULTS USING HIGHEST                                      NORMAL AS CUTOFF (NORMALS = 10)                                                               POSITIVES   POSTIVES FROM                                     SAMPLE TYPE     N  FROM TGF TGF/EGF RATIO                                     __________________________________________________________________________    BREAST          3  0/3  (0%)                                                                              1/3 (33%)                                         MYELOMA         14  7/14                                                                             (50%)                                                                               8/14                                                                             (57%)                                         PROSTATE (PROGRESSIVE)                                                                        7  3/7 (43%)                                                                              5/7 (71%)                                         PROSTATE (STABLE)                                                                             8  1/8 (12%)                                                                              1/8 (12%)                                         PROSTATE (UNRATED)                                                                            2  0/2  (0%)                                                                              0/2  (0%)                                         RECTAL          1  1/1 (100%)                                                                             1/1 (100%)                                        __________________________________________________________________________

EXAMPLE VII USE OF MULTIPLE-LAYER CUSHIONS

Different materials of potential use as primary or secondary cushionlayers were tested for their ability to maintain discrete boundariesduring formation of the cushion and subsequent centrifugation, and toallow the pelleting of S. aureus particles in a brief spin. Allpotential cushion materials were tested for the ability of fixed S.aureus to pellet in 0.4 mL polypropylene microcentrifuge tubes during aone minute centrifugation at full speed in a microcentrifuge (Savant,10,000 RPM). Under these conditions, pelleting occurred equally well forsucrose solutions (10-40% w/v, d=1.0374-1.1758 at 22° C.) and thewater-immiscible materials listed below: diethyl succinate, ethylcinnamate, dibutyl phthalate, methyl adipate, and diethyl maleate.

EXAMPLE VIII COMPETITIVE RIA FOR STIMULATIONG HORMONE (TSH)

A commercial 125-iodine RIA kit for determining TSH was obtained fromAmerican Bioclinical (Portland, Oreg.) and adapted to the separation anddetection methods of the present invention. All assay reactants wereused according to the manufacturer's instructions except that reactantvolumes were decreased four-fold, and S. aureus (25 μL of a 10% w/vsuspension per test) was substituted for the "second antibody"precipitating solution. The adapted test was performed using 0.4 mLmicrocentrifuge tubes containing 0.25 mL butyl phthalate cushions.

Even though the adapted test was only incubated for two hours (37° C.)versus four hours (25° C.) for the standard test, the adapted testexhibited significantly lower nonspecific binding with equivalent totalbound and greater overall sensitivity. Detailed results are given below:

    __________________________________________________________________________    COMPARISON OF STANDARD RIA AND ADAPTED TSH RIA                                CONDITIONS      STANDARD TEST  ADAPTED TEST                                   __________________________________________________________________________    TIME:           4 HOURS        2 HOURS                                        TEMPERATURE:    ROOM TEMPERATURE                                                                             37° C.                                  ASSAY MIXTURE VOLUME:                                                                         0.50 ML + 1 ML .15 ML                                                         PRECIPITATING SOLN                                            USER STEPS:     1. MIX SAMPLE +                                                                              1. MIX SAMPLE +                                                 ANTIBODY       ANTIBODY + TRACER                                             2. ADD TRACER  2. SPIN 0.5 MINUTE                                             3. ADD 2ND ANTIBODY                                                                          3. COUNT CPM                                                   4. SPIN 10 MINUTES                                                            5. DRAIN SUPERNATANT                                                          6. COUNT CPM                                                  __________________________________________________________________________    RESULTS                                                                                      STANDARD TEST                                                                            ADAPTED TEST                                        SAMPLE         % BOUND    % BOUND                                             __________________________________________________________________________    TOTAL CPM ADDED                                                                              N.A.       N.A.                                                NONSPECIFIC    3.9%       1.7%                                                BINDING (NRS)                                                                 TOTAL BOUND    37.6%      37.2%                                               25 μU/mL in RIA                                                                           15.2%      N.A.                                                33 μU/mL in RIA                                                                           12.0%      7.2%                                                50 μU/mL in RIA                                                                           7.9%       N.A.                                                __________________________________________________________________________

EXAMPLE IX PERFORMANCE OF RIA COMPONENTS: PRECISION & SHIELDINGEFFICIENCY

(A) Shielding effectiveness of radiation shields.

Radiation shields (42, FIG. 3) were tested for efficiency of shielding125-iodine radiation, with and without shielding disks 52. Aliquots of125-I containing solutions were pipetted into 0.4 mL assay vessels.Total unshielded counts were determined using tubes without cushions,counted without shields. Detection efficiency was determined by countingthese same tubes in the two types of shields. Shielding efficiency wasdetermined by counting tubes containing cushions with two kinds ofshields (FIG. 3, with and without the disk 52).

    ______________________________________                                        DETECTION OF BOUND LABEL                                                                          DETECTION OF                                              (IN DISTAL END      UNBOUND (IN ASSAY                                         OF ASSAY VESSEL)    MIXTURE)                                                  CPM              SHIELD            SHIELD                                     ADDED   SHIELD   WITH DISK  SHIELD WITH DISK                                  ______________________________________                                        2687    103%     94%        -0.1%  0.4%                                       4921    103%     94%        0.2%   0                                          7407    103%     97%        0.2%   0.2%                                       9620     98%     93%        0.1%   0.1%                                       12494    97%     94%        0      0.1%                                       15379    96%     89%        0.1%   0                                          ______________________________________                                    

(B) Precision for RIA

Total bound tracer replicates were measured using the TGF assay (ExampleIA). Four groups of 15 tubes each were counted on two different gammacounters.

    ______________________________________                                        MICROMEDIC FOUR-CHANNEL COUNTER                                               (3 MINUTE COUNTS)                                                             SAMPLE SET AVERAGE    STD DEVIATION % CV                                      ______________________________________                                        I          2222       69            3.1                                       II         2121       85            4.0                                       III        2114       89            4.2                                       IV         2113       104           4.9                                       ______________________________________                                    

    ______________________________________                                        BECKMAN ONE-CHANNEL COUNTER                                                   (ONE MINUTE COUNTS)                                                           SAMPLE SET AVERAGE    STD DEVIATION % CV                                      ______________________________________                                        I          2265       90            4.0                                       II         2194       96            4.0                                       III        2170       97            4.5                                       IV         2152       123           5.7                                       ______________________________________                                    

EXAMPLE X RADIOIMMUNOASSAY FOR DIGOXIN

A rapid assay using the present invention was compared with conventionaldouble antibody assay methods. Commercially-available preparations ofrabbit antiserum (Immunosearch, San Francisco, Calif.), and 125-Iodinelabelled digoxin and digoxin standards (from Cambridge MedicalDiagnostics, Cambridge, Mass.), were used. The binding component wasprepared as a 10:1 blend of 10% Tachisorb™ with 10% Woods strain of S.Aureus (both from Behring Diagnostics, La Jolla, Calif.). Theanti-digoxin antibody concentration in each assay type was adjusted tobind approximately 60% of 50,000 CPM of labelled digoxin after a fifteenminute incubation. Centrifugation was for five minutes at ca. 10,000×gin a Savant microcentrifuge with a 36-tube, fixed angle rotor.Radioactivity was determined with one minute counts.

Method: To 0.4 mL polypropylene tubes containing 0.200 mL dibutylphthalate was added 50 microliters containing labelled digoxin, 1% NGS,5% w/v Tachisorb-R™ and 0.5% Sansorbin™ (Behring Diagnostics, San Diego,Calif.), and 1% NP-40 (Sigma Chemicals, St. Louis Mo.). A 50 microlitersample was added to each tube, then the assay was initiated by rapidlyadding 50 microliters of antiserum diluted in 1% NGS. Tubes were cappedand mixed by partially inverting several times, then incubatedstationary for 15 minutes.

After centrifugation, assay tubes were transferred to radiation shields(Biotope Cat #AC-010, essentially as described in FIG. 3) andradioactivity determined in the gamma counter. Results are describedbelow

    ______________________________________                                        Results:                                                                      ______________________________________                                        PRECISION                                                                     N =           Mean      % CV                                                  ______________________________________                                        18            1.57 ng/mL                                                                              4.89                                                  18            0.68 ng/mL                                                                              3.65                                                  SENSITIVITY                                                                   .07 ng/mL (2 s.d. from "zero")                                                ______________________________________                                        CORRELATION WITH COMMERCIAL DIGOXIN ASSAYS:                                   (42 patient samples)                                                          ______________________________________                                        versus Clinical Assays RIA                                                             R = 0.957748                                                                  slope = .953                                                                  intercept = 0.119 ng/mL                                              versus Cambridge Medical Diagnostics RIA                                               R = 0.977                                                                     slope = 1.014                                                                 intercept = -.029 ng/mL                                              ______________________________________                                    

These results are striking in that the assay of the present invention iscompleted in less than twenty minutes, yet correlates well with, andprovides better precision than current clinical assays which take 1-2hours to complete.

EXAMPLE XI ENZYMEIMMUNOASSAY FOR DIGOXIN

An enzyme-labelled digoxin assay using the method of the presentinvention is adapted from a commercial digoxin kit (Immunotech, Allston,Mass.). The only changes in reagents are the inclusion of 20% sorbitolin the color developer solution, and the substitution of 50 microlitersof Tachisorb-R (Behring Diagnostics, La Jolla, Calif.) for the 500microliters of precipitating solution (goat-anti rabbit IgG) providedwith the kit.

In two mL, screw-top microcentrifuge tubes (Sarstedt, Princeton, N.J.),1 mL of sorbitol-color developer is dispensed, then overlaid with 0.4 mLof dibutyl phthalate. A 200 microliter assay mixture is prepared byadding 100 microliters of Tachisorb-R, 25 microliters of digoxin-enzyme(alkaline phosphatase) conjugate, 25 microliters of sample (serum-basedstandards), and 50 microliters of antibody solution. The assay isincubated at room temperature for 15 minutes, centrifuged two minutes at10,000×g, and further incubated one hour for color development.

Color is measured at 400 nm in a Shimadzu Model UV-160spectrophotometer. Because of the opacity of the polypropylene tubes,results for each tube are corrected for nonspecific absorbance at 500nm. Results are shown below:

    ______________________________________                                                    ABSORBANCE AT 400 nm                                              SAMPLE      (corrected)                                                       ______________________________________                                        0           180                                                               1.0         150                                                               2.0         100                                                               4.0          80                                                               ______________________________________                                    

In this assay, normal rabbit serum is present as a diluent for theanti-digoxin antibody, and the total IgG exceeds the capacity of thebinding components added. Substantially greater signal can be obtainedby optimizing the method to capture all of the analyte-specific antibodyin the assay.

EXAMPLE XII SEMIQUANTITATIVE VISUAL ASSAY FOR DIGOXIN

The enzyme-labeled cushion assay adapted from the Immunotech digoxinassay was performed as described in Example XI, except that 0.4 mL assaytubes were used, with 100 microliters each of color developer anddibutyl phthalate, 25 microliters of Tachisorb-R, 25 microliters ofdigoxin-enzyme conjugate, 25 microliter samples, and 50 microliters ofantibody solution.

After 15 minutes at room temperature, tubes were centrifuged at 10,000×gfor one minute. Color initially developed in the particle pellets andgradually migrated in the liquid color developer layer. Low standards(0,1,2 ng/mL) could be distinguished visually from high standards (4,8ng/mL) after a ten minute color development at room temperature (22degrees C.).

EXAMPLE XIII AFFINITY BINDING ASSAY FOR GLYCOSYLATED HEMOGLOBIN

The principle of the test is that glycosylated hemoglobin binds to any"affinity resin", boronic acid particles (Glyco-Gel B™) which wereobtained from the Pierce Chemical Company (Rockford, Ill.). These wereused as binding components in a non-immunological assay of the presentinvention. After centrifuging the binding components through a primarylayer, bound hemoglobin was eluted with a sugar solution (15% sorbitol)contained in a secondary layer. The color in the secondary layer wasmeasured with a spectrophotometer to provide quantified results. If thesorbitol solution was omitted, visual detection of hemoglobin bound tothe solid phase was possible.

Method: Into a two mL microcentrifuge tube was pipetted 0.7 mL 15%sorbitol, then 0.3 mL primary layer material (diethylmethylmalonate),followed by 0.3 mL of a 25% (v/v) aqueous suspension of bindingcomponents. To begin the assay, 0.05 mL of hemolysed blood (1:10dilution) was added to the particle suspension and incubated 30 minutesat room temperature or 10 minutes at 37° C. After centrifugation at5000×g for five minutes, absorbance at 418 nm was determined using theassay vessel as a cuvette. Nonspecific absorbance at 500 nm wassubtracted. Total hemoglobin or non-glycosylated hemoglobin wasdetermined separately and the % glycosylated was calculated.

Results: Normal and elevated standards provided with a Pierce clinicaldiagnostics kit for glycosylated hemoglobin were consistentlydistinguished using both quantitative and visual detection methods.Essentially all of the bound hemoglobin is released from the particlesinto the sorbitol secondary layer, allowing for accurate quantitation.

I claim:
 1. An assay vessel for separating bound label from unboundlabel within an assay mixture, wherein said assay mixture includes oneor more binding components, label bound to at least some of said bindingcomponents, and a substantially aqueous solution containing unboundlabel, said binding components differing in apparent density from saidaqueous solution, comprising:a vessel having a proximal end and a closeddistal end, said vessel defining an elongated chamber therein; a primarylayer extending generally transversely within the chamber to form aselective barrier therein, said primary layer being immiscible with bothsaid unbound label and said binding components; and a barrier layerbetween said primary layer and said proximal end.
 2. The assay vessel ofclaim 1 wherein said primary layer is selectively liquifiable.
 3. Theassay vessel of claim 1 wherein said primary layer is a mixture of twoor more water-immiscible substances that are miscible with one another.4. The assay vessel of claim 3 wherein at least one of saidwater-immiscible substances is selectively liquifiable.
 5. The assayvessel of claim 1 wherein said primary layer is a mixture of two or morewater-immiscible substances, at least one of which has a greater densitythan water when in liquid form.
 6. The assay vessel of claim 1 whereinsaid barrier layer is selectively liquifiable and is substantiallymiscible with the assay mixture.
 7. The assay vessel of claim 6 whereinsaid barrier layer is a gel layer of agarose or collagen.
 8. The assayvessel of claim 1, further including a reagent reservoir, said reservoircontaining additional reaction components.
 9. The assay vessel of claim8 wherein said reagent reservoir is positioned between said primarylayer and said distal end.
 10. The assay vessel of claim 8 wherein saidreservoir is positioned adjacent to said proximal end, said reservoirfurther including a reversibly sealed aperture.
 11. The assay vessel ofclaim 1, further including one or more secondary layers positionedbetween said primary layer and said distal end.
 12. The assay vessel ofclaim 1 wherein said primary layer is of a different density than saidaqueous solution.
 13. An assay vessel for separating bound label fromunbound label within an assay mixture, wherein said assay mixtureincludes one or more binding components, label bound to at least some ofsaid binding components, and a substantially aqueous solution containingunbound label, said binding components differing in apparent densityfrom said aqueous solution, comprising:a vessel having a proximal endand a closed distal end, said vessel defining an elongated chambertherein; and a selectively liquifiable primary layer extending generallytransversely within the chamber to form a selective barrier therein,said primary layer being immiscible with both said unbound label andsaid binding components, and of different density than said bindingcomponents.
 14. A reusable detection vessel for use in specific bindingassays, comprising:an elongated container having an open proximal endand a closed distal end; a radiation-absorbing shield adapted to fitwithin said container, said shield positioned therein to provide ashielded portion and an unshielded portion toward said closed end; andan assay vessel having a proximal end and a closed distal end, saidassay vessel defining an elongated chamber therein and adapted to beslidably received by said shield, said assay vessel further including aprimary layer extending generally transversely within the chamber toform a selective barrier therein.